Two calcium-activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187.

1. Currents evoked by elevated intracellular free Ca2+ in Xenopus laevis oocytes were studied using the two-electrode voltage clamp technique. The elevation in Ca2+ concentration was achieved in three ways: by the use of the divalent cation ionophore A23187; by application of Ca2+-mobilizing neurotransmitters serotonin and acetylcholine (ACh); by the entry of Ca2+ through voltage-dependent channels. 2. In most experiments, the membrane was permeabilized to Ca2+ by a 15 min pretreatment with A23187 in a Ca2+-free solution. Exposure of the ionophore-treated oocytes to external Ca2+ elicited an inward current (at holding potentials of -40 to -60 mV). At external Ca2+ concentrations ([Ca2+]) between 0.1 and 1 mM, the current had a time-to-peak of at least 10 s, and slowly decayed over tens of seconds. At [Ca2+] greater than 2 mM, the inward current had two distinct kinetic components, a fast and transient one (Ifast) and a slow one (Islow). 3. The main carrier of the Ca2+-evoked inward current was Cl-. Several data indicate the existence of a tetraethylammonium (TEA)-sensitive K+ conductance. No evidence for a Na+ current was found. 4. The two components of the Ca2+-evoked inward current in ionophore-permeabilized oocytes, and the two components of the current evoked by ACh and serotonin (the latter in oocytes injected with rat brain RNA but untreated with A23187), were blocked by intracellular injection of the Ca2+ chelator, ethyleneglycolbis-(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA). The two components of these currents displayed different sensitivity to Ca2+ buffering; higher doses of EGTA were necessary to inhibit the slow component than the fast one. 5. One to two minutes of treatment with 2 mM-9-anthracene carboxylic acid (9-AC) fully blocked Ca2+-dependent Cl- current evoked by Ca2+ influx through voltage- dependent Ca2+ channels in intact (untreated with A23187) oocytes. In ionophore-treated oocytes, block of Ifast was observed at holding potentials at which the current was outward (i.e. due to Cl- influx); Islow was inhibited only partially. The block of Ca2+-evoked Cl- efflux by 9-AC developed much more slowly and was less potent. to explain these results, the existence of two sites of 9-AC action is proposed. 6. Exposure of the ionophore-permeabilized oocytes to 0.1-0.2 mM [Ca2+] strongly reduced the response to higher concentrations of Ca2+. Ifast displayed stronger Ca2+-dependent inactivation than Islow.(ABSTRACT TRUNCATED AT 400 WORDS)

Utility of the signal-averaged electrocardiogram in patients presenting with sustained ventricular tachycardia or fibrillation while on an antiarrhythmic drug.

The utility of the signal-averaged electrocardiogram (SAECG) for predicting ventricular tachycardia (VT) induction in patients presenting with sustained VT or ventricular fibrillation (VF) while on an empirically chosen antiarrhythmic agent was assessed in 17 patients. At the time of presentation with a malignant arrhythmia, 12 patients were taking quinidine, three patients were taking procainamide, and two patients were taking flecainide. All patients underwent programmed ventricular stimulation when not taking antiarrhythmic drugs; 12 patients had no inducible sustained VT and five patients had inducible sustained monomorphic VT. The SAECG done in the control state without antiarrhythmic agents was negative for late potentials in 11 of 12 patients in the noninducible group and positive for late potentials in four of five patients in the inducible group (sensitivity = 80% and specificity = 92%). We conclude that in patients presenting with life-threatening ventricular arrhythmias while taking an antiarrhythmic drug, the SAECG distinguishes patients with possible proarrhythmic events from those who have the substrate for inducible sustained VT.

The involvement of inositol 1,4,5-trisphosphate and calcium in the two-component response to acetylcholine in Xenopus oocytes.

1. The membrane response to acetylcholine (ACh), inositol 1,4,5-trisphosphate (IP3) and intracellular Ca2+ was studied in Xenopus laevis oocytes under voltage-clamp conditions. 2. Shallow, submembranal injections of IP3 in the animal hemisphere of the oocyte evoked a two-component response comprised of a rapid, transient component followed by a slow, sustained component. 3. When the injection pipette was inserted further into the cell (to 300 microns below the cell membrane), the fast component diminished and the slow component remained unchanged or even increased. 4. The rapid component exhibited an apparent higher sensitivity to IP3 compared to the slow component. 5. The two components of the IP3 response were retained in a Ca2+-free environment. 6. Injection of a single large dose (20-50 pmol) of CaCl2 into the oocyte evoked a typical two-component response, whereas repetitive threshold doses (0.1 pmol CaCl2) elicited large current fluctuations which developed into a small depolarization current. 7. The delay in the peak of the slow component of the response to either IP3 or to CaCl2 injections appeared too long to be accounted for by diffusion alone. 8. Depletion of oocyte Ca2+ by the divalent cation ionophore A23187 (greater than 1 microM) inhibited the response to ACh and IP3. Low concentrations of A23187 selectively inhibited the rapid component of the ACh response, though not the rapid component of the IP3 response. 9. Our data suggest that the two-component membrane response to ACh in Xenopus oocytes can be accounted for by ACh-induced elevation of IP3 and subsequent IP3-induced release of intracellular Ca2+.

Dissociation of acetylcholine- and cyclic GMP-induced currents in Xenopus oocytes.

In Xenopus follicular oocytes, activation of muscarinic receptors evokes a slow potassium current (H-response); a similar current is evoked by intracellular injection of cyclic guanosine 3',5'-monophosphate, cGMP (Dascal et al. 1984). We have tested the hypothesis that cGMP may be the second messenger that mediates the opening of K channel by acetylcholine (ACh). ACh elevated the intracellular level of cGMP with a time course similar to that of the development of the muscarinic H-response; maximal increase in cGMP concentration above the control was about 0.2 pmole/oocyte. The amount of injected cGMP that produced a detectable K current ("threshold dose") varied between 0.5 and 3 pmole/oocyte. At low doses of cGMP, the slope of log dose-log response curve was about 2.5, suggesting involvement of a biochemical process with a positive cooperativity of at least 3. Higher doses of cGMP evoked, in addition to the outward current, an irregular, rapidly developing, long-lasting inward current, that never reached amplitudes comparable to those of ACh-evoked Cl currents. The K current elicited by cGMP was insensitive to elevation or depletion of external Ca. It was potentiated by isobutylmethylxanthine (IBMX). ACh strongly inhibited the cGMP-evoked K current when applied at the plateau of the latter. 4-Phorbol 12,13-dibutyrate (PDBu) (1 microM) rapidly and completely inhibited the cGMP response. It is concluded, that most of the results presented in this report contradict the hypothesis that cGMP is the intracellular mediator of ACh-induced changes in membrane conductance in the oocytes.

The signal-averaged electrocardiogram as a screening test for inducibility of sustained ventricular tachycardia in high risk patients: a prospective study.

The role of the signal-averaged electrocardiogram in predicting the induction of sustained monomorphic ventricular tachycardia in high risk patients was assessed prospectively in 100 consecutive patients. Presenting diagnoses were syncope (38 patients), nonsustained ventricular tachycardia (24 patients), sustained ventricular tachycardia (25 patients) and sudden cardiac arrest (13 patients). Using programmed ventricular stimulation, 71 patients (group I) did not have and 29 patients (group II) did have inducible sustained monomorphic ventricular tachycardia. Using the signal-averaged electrocardiogram with filtering (6 dB/octave) at high pass corner frequencies of 67 and 100 Hz, the two groups were compared. The signal-averaged electrocardiogram was considered abnormal if all of the following criteria were satisfied: 1) the total filtered QRS complex duration was greater than 120 ms, 2) the duration of the terminal QRS complex of less than or equal to 20 microV was greater than or equal to 30 ms, and 3) at least one deflection (late potential) was present in this region. Differences between groups I and II in these three measures were highly significant (p less than or equal to 0.001). The sensitivity and specificity of signal averaging for predicting the induction of sustained ventricular tachycardia were 93 and 94%, respectively. Stepwise logistic regression analysis identified the signal-averaged electrocardiogram as the best predictor of induction of sustained monomorphic ventricular tachycardia, independent of left ventricular ejection fraction, presence of ventricular aneurysm, myocardial infarction and other clinical variables (chi-square = 93.2, p less than 0.0001). The signal-averaged electrocardiogram is a sensitive and specific test for the induction of sustained monomorphic ventricular tachycardia, having independent predictive value.

Detection of late potentials on the surface electrocardiogram in unexplained syncope.

To assess the usefulness of signal averaging of the surface electrocardiogram for detecting hitherto undocumented ventricular tachycardia (VT) in patients with unexplained syncope, 24 such patients were evaluated by electrocardiography and programmed ventricular stimulation. The surface electrocardiograms of 15 normal volunteers and 22 patients with documented sustained VT were also examined. No study subject had a bundle branch block or a QRS duration longer than 120 ms. Sustained VT was recorded in 9 of the 24 patients with syncope (8 patients with inducible VT and 1 with a spontaneous episode of recorded sustained VT). The signal-processed electrocardiogram contained late potentials and a filtered QRS duration longer than 120 ms in 8 of these 9 patients (89% sensitivity). None of the remaining 15 patients had these electrocardiographic abnormalities. Similar results were found in the patients with previously documented sustained VT (82% sensitivity) and in normal volunteers (no instances of abnormal recordings). In patients with unexplained syncope, signal processing of the surface electrocardiogram may be a sensitive and specific noninvasive test for detecting a high-risk subset of patients prone to lethal ventricular tachyarrhythmias.

Acetylcholine- and inositol 1,4,5-trisphosphate-induced calcium mobilization in Xenopus laevis oocytes.

Acetylcholine induces a complex electrical membrane response in Xenopus laevis oocytes. This response is mimicked, and probably mediated by injected inositol 1,4,5-trisphosphate. Oocytes prelabelled with 45Ca released calcium in two phases, the second, slow phase exhibiting first order kinetics of release. Brief exposure of prelabelled oocytes to acetylcholine resulted in a significant increase in the rate of calcium release that returned to control values 2-3 min following the removal of the neurotransmitter. Intracellular injection of inositol 1,4,5-trisphosphate resulted in increased rate of calcium release similar to, but longer than that caused by acetylcholine. Experiments conducted on single oocytes permitted the investigation of the relationship between acetylcholine-induced and inositol 1,4,5-trisphosphate-induced calcium mobilization and the resulting electrical membrane response. Our data reinforce our previous suggestion that inositol 1,4,5-trisphosphate is the intracellular second messenger of the muscarinic membrane electrical response in Xenopus oocytes.

ATP-evoked membrane responses in Xenopus oocytes.

Voltage-clamp technique and intracellular injections of drugs were used to study the adenosine triphosphate (ATP)-evoked depolarizing current response in the Xenopus laevis oocytes. The depolarizing current was comprised of a fast transient component (D1) followed by a late long-lasting component (D2). It was carried mainly by Cl- ions. The depolarizing current was better elicited by ATP and ADP than by AMP or adenosine and was not blocked either by theophylline (0.2 mM) or by quinidine sulphate (1 mM). The D2 current was sometimes masked by an ATP-evoked K+ hyperpolarizing current which was blocked by theophylline and mediated via P1 purinoceptors. This study suggests that the oocyte's membrane embodies at least two different purinoceptor's types, each of these types subserves a different set of ionic channels.

Role of calcium mobilization in mediation of acetylcholine-evoked chloride currents in Xenopus laevis oocytes.

The involvement of Ca ions in the mediation of muscarinic Cl- current responses in Xenopus oocytes was studied using the voltage-clamp technique and direct measurements of 45Ca efflux. The injection of Ca into the oocytes produced a dose-dependent transient inward (depolarizing) current carried by Cl. This current was occasionally followed by a second, long-lasting inward current. The muscarinic response was evoked by the application of acetylcholine (ACh). It consisted of a transient inward current response, and a long-lasting inward current response, both inward currents carried by Cl ions. Both responses were inhibited by intracellular injection of ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), the long-lasting response being inhibited faster than the transient response. The calmodulin inhibitor, trifluoperazine, inhibited both the Cl-current responses to ACh and to Ca injection. ACh (10 microM) evoked a release of 45Ca from pre-loaded oocytes. This effect was inhibited by atropine (1 microM). In the absence of external Ca, the muscarinic transient and long-lasting responses were partially inhibited. The long-lasting response was more sensitive to the external Ca depletion than the transient response. Repetitive applications of ACh in the absence of external Ca resulted in a progressive decrease in the response amplitudes. Under these conditions, a temporary exposure to normal Ca solution ('Ca window') resulted in a partial recovery of the response amplitudes. The muscarinic inward current responses were not inhibited by nifedipine (20 microM). In the presence of a high external concentration of Mn ions ([Mn]o = 18 mM), the transient response was potentiated. Subsequent applications of ACh in high [Mn]o resulted in progressively decreasing responses. It is concluded that the muscarinic Cl responses in Xenopus oocytes are mediated by an increase in the intracellular free Ca activity, aiCa. Ca ions involved in the mediation of the muscarinic Cl current responses are released from cellular Ca stores. It is also proposed that the transient and long-lasting responses result from the release of Ca from two different stores.

Acetylcholine and phorbol esters inhibit potassium currents evoked by adenosine and cAMP in Xenopus oocytes.

In Xenopus laevis oocytes, adenosine and other purinergic agonists induce a K+-conductance increase that is fully mimicked by intracellular application of cAMP. Acetylcholine suppresses the K+-conductance increase caused by adenosine, by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or by intracellular injection of cAMP. This effect of acetylcholine is not mimicked by intracellular injection of Ca2+ or of the Ca-mobilizing agent inositol 1,4,5-trisphosphate. However, adenosine and cAMP responses are inhibited by 4 beta-phorbol 12,13-dibutyrate and 4 beta-phorbol 12-myristate 13-acetate. These results suggest that, in Xenopus oocytes, the muscarinic inhibition of purinergic and cAMP responses is mediated through the activation of the phospholipid-dependent, Ca-activated protein kinase (protein kinase C).

Adenosine-induced K+ current in Xenopus oocyte and the role of adenosine 3',5'-monophosphate.

Voltage clamp technique was used in Xenopus laevis oocytes in order to study and compare membrane currents evoked by extracellularly applied adenosine (0.1-10 microM) and intracellularly injected cyclic AMP (0.15-10 microM). The adenosine response is a late long-lasting outward K+ current ("H" current), mediated by the Ra purine receptor subtype. The H current amplitude is directly proportional to (occupancy)3; the KD for adenosine is 3.34 microM. The H current is inhibited by the intracellular injection of protein kinase inhibitors, types II and III (5-450 ng/oocyte) and is usually potentiated by intracellular injection of theophylline (100-300 microM), though extracellular application of theophylline (1-100 microM) reversibly blocks the receptor. Occasionally, the H current is contaminated by a small Cl- current. The cyclic AMP current is also a long-lasting K+ outward current which is potentiated by extracellular theophylline (2 mM). Injection of cyclic AMP inhibits the membrane response to subsequent application of adenosine. The converse inhibition of a cyclic AMP response by an earlier adenosine response is also observed but at very high concentrations of adenosine (greater than 0.6 mM). It was shown by radioimmunoassay that extracellular adenosine increases the level of the intracellular cAMP within a few seconds by about 30%. Intracellular injection of a comparable amount of cAMP was shown to evoke a measurable K+ current. It is proposed that the adenosine-evoked K+ outward current is mediated by a rise in intracellular cAMP.

The mechanism of steroid anaesthetic (alphaxalone) block of acetylcholine-induced ionic currents.

The effects of the steroid anaesthetic alphaxalone on acetylcholine (ACh)-induced ionic channels were studied in voltage clamped 'myoballs' in culture. Alphaxalone produced a reversible blockade of the ACh-evoked inward current, ED50 = 6.0 microM. The ACh reversal potential (-5.0 mV), the single channel conductance (13.5 pS) and mean open time (3.6 ms) were unchanged by the drug. Thus, alphaxalone produced an 'all or none' block of the ionic channel. In double pulse conditioning experiments, alphaxalone produced an additional inhibition with a time constant of recovery (550 ms) much longer than the time constant of recovery of the normal desensitization (250 ms). It was concluded that alphaxalone blocks active (open) ionic channels.

Xenopus oocyte resting potential, muscarinic responses and the role of calcium and guanosine 3',5'-cyclic monophosphate.

Resting potential (r.p.) and muscarinic response mechanisms were studied in Xenopus laevis oocytes using the voltage-clamp technique. Insertion of micro-electrodes into the oocyte produced a 'shunt' membrane conductance which partially sealed after a few minutes. The oocyte resting potential (measured with a single intracellular electrode) ranged from -40 to -60 mV. Ouabain and low K+ solution depolarized both follicles and denuded oocytes. The electrogenic Na+-K+ pump was more active in the latter. In the presence of ouabain, the r.p. agreed with the constant field theory. alpha (PNa+/PK+) was 0.12 in follicles and 0.24 in denuded oocytes. beta (PCl-/PK+) was 0.4 in both. At [Na+]o lower than 70 mM, the r.p. deviated considerably from the constant field predictions. The relatively large value of alpha indicated the major role of Na+ in oocyte r.p. determination. The oocyte muscarinic response was separated into four distinct components: the fast depolarizing Cl- current, 'D1'; the slow depolarizing Cl- current, 'D2'; the slow hyperpolarizing K+ current, 'H'; and the large membrane Cl- current fluctuation, 'F'. The H response reversal potential showed a Nernst relationship to [K+] and was selectively blocked by intracellular injection of tetraethylammonium (TEA). The D1 and D2 reversal potential showed a Nernst relationship to [Cl-]. In Ca2+-deficient, EGTA-containing medium, D2 and F were abolished and D1 and H were reduced. Verapamil inhibited all responses. Increasing [Ca2+]o caused a significant increase in D1, D2 and F response amplitudes. Intracellular injection of 0.6-10 pmol guanosine 3',5'-cyclic monophosphate, induced a large outward K+ current, similar to the muscarinic H response.

Evidence for acetylcholine receptor blockade by intracellular hydrogen ions in cultured chick myoballs.

Acetylcholine (ACh)-induced membrane currents were recorded in voltage-clamped myoballs in culture. The ACh sensitivity and the charge transfer per channel (Qc) were doubled in NH4+ at normal extracellular pH (pHo). Immediately after wash-out of NH4+, the ACh sensitivity and Qc undershot below the control. The recovery period of the ACh sensitivity and Qc was about 20 min. The ACh sensitivity and Qc greatly increased at alkaline pHo and decreased at acid pHo. We suggest that the intracellular H+ ions can block the ACh-induced channel. The pHo effects may be mediated by a similar (but smaller) change in the intracellular pH (pHi). Intracellular alkalinization can improve the channel function.

Monoclonal antibodies modify acetylcholine-induced ionic channel properties in cultured chick myoballs.

Monoclonal antibodies directed against the cholinergic binding site of the acetylcholine receptor were found to alter the ion channel properties in cultured chick "myoballs." Time and dose dependent reduction in acetylcholine sensitivity was observed. Noise analysis experiments indicated a decrease in the mean single channel conductance and an increase in the mean single channel open time.