Dalia Varon

Gαi Controls the Gating of the G Protein-Activated K+ Channel, GIRK

GIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of G(beta)(gamma). The role of G(alpha) subunits in GIRK gating is elusive. Here we demonstrate that G(alpha)(i) is not only a donor of G(beta)(gamma) but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or G(beta)(gamma). Coexpression of G(alpha)(i3) or G(alpha)(i1) restores the correct gating parameters. G(alpha)(i) acts neither as a pure G(beta)(gamma) scavenger nor as an allosteric cofactor for G(beta)(gamma). It inhibits only the basal activity without interfering with G(beta)(gamma)-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. G(alpha)(i) probably acts in its GDP bound form, alone or as a part of G(alpha)(beta)(gamma) heterotrimer.

Gβγ-dependent and Gβγ-independent Basal Activity of G Protein-activated K+ Channels

Cardiac and neuronal G protein-activated K+ channels (GIRK; Kir3) open following the binding of Gβγ subunits, released from Gi/o proteins activated by neurotransmitters. GIRKs also possess basal activity contributing to the resting potential in neurons. It appears to depend largely on free Gβγ, but a Gβγ-independent component has also been envisaged. We investigated Gβγ dependence of the basal GIRK activity (AGIRK,basal) quantitatively, by titrated expression of Gβγ scavengers, in Xenopus oocytes expressing GIRK1/2 channels and muscarinic m2 receptors. The widely used Gβγ scavenger, myristoylated C terminus of β-adrenergic kinase (m-cβARK), reduced AGIRK,basal by 70–80% and eliminated the acetylcholine-evoked current (IACh). However, we found that m-cβARK directly binds to GIRK, complicating the interpretation of physiological data. Among several newly constructed Gβγ scavengers, phosducin with an added myristoylation signal (m-phosducin) was most efficient in reducing GIRK currents. m-phosducin relocated to the membrane fraction and did not bind GIRK. Titrated expression of m-phosducin caused a reduction of AGIRK,basal by up to 90%. Expression of GIRK was accompanied by an increase in the level of Gβγ and Gα in the plasma membrane, supporting the existence of preformed complexes of GIRK with G protein subunits. Increased expression of Gβγ and its constitutive association with GIRK may underlie the excessively high AGIRK,basal observed at high expression levels of GIRK. Only 10–15% of AGIRK,basal persisted upon expression of both m-phosducin and cβARK. These results demonstrate that a major part of Ibasal is Gβγ-dependent at all levels of channel expression, and only a small fraction (<10%) may be Gβγ-independent.

A new class of antiarrhythmic-defibrillatory agents

Novel dibenzoazepine and 11-oxo-dibenzodiazepine derivatives are shown to be effective ventricular defibrillating drug candidates. They exhibit significant in vivo defibrillatory activity with no observed changes in ECG either before or after the VF event. These compounds also exhibit antifibrillatory activity by elevating the fibrillation threshold potential, all suggesting that such drugs could be used to treat VF either by themselves or together with electrical defibrillators.

The protective effect of class III antiarrhythmic agents against calcium overload in cultured myocytes

Calcium ions have been implicated in the mechanisms of ventricular arrhythmias. Impairment of intercellular coupling by calcium overload is considered to facilitate ventricular fibrillation (VF) and to sup-press its self termination. According to our hypothesis, any compound that decreases intracellular calcium concentration [Ca2+]i during VF can serve as defibrillating drug. In this study, we examined the effect of d-sotalol and tedisamil on calcium overload in cultured, spontaneously beating rat cardiomyocytes. The changes of [Ca2+]i were measured by indo-1 method and the intercellular synchronization by image analysis. The results showed that increase in [Ca2+]o from 1.9 mM to 3.9 mM increased [Ca2+]i from 100 nM to 320 nM and transformed the synchronized cell movement to an asynchronous one. Administration of 5 x 10(-6) M d-sotalol or 10(-6) M tedisamil, decreased the [Ca2+]i to its basic level and restored the synchronized activity. In summary: Our results showed that increase in [Ca2+]i known to cause inhibition of intercellular coupling, that could lead to arrhythmia and fibrillation while d-sotalol or tedisamil prevented this effect. These results support our hypothesis, that class III antiarrhythmic compounds with positive inotropic effect, increase intercellular synchronization, by decreasing free [Ca2+]i, most probably by increasing the Ca2+ uptake by the sarcoplasmic reticulum, and therefore act as a defibrillating compound.

Structural Substrates Involved in the Development of Severe Arrhythmias in Hypertensive Rat and Aged Guinea Pig Hearts

We hypothesize that age- as well as hypertension-related myocardial remodeling can deteriorate cell-to-cell junctions and communication, thus consequently facilitate re-entry arrhythmias. The aim of the study was to characterize structural substrate that precede appearance of atrial fibrillation in aged guinea pig heart and occurrence of ventricular fibrillation in hypertensive rat heart. The experiments were performed on Langendorff-perfused heart. To induce atrial fibrillation the left atrium of old or young guinea pig was stimulated by 1 sec burst of 0.1 msec rectangular pulses at 50–70 pps. As soon as sinus rhythm was detected the stimuli burst was delivered again. To induce ventricular fibrillation the heart of hypertensive or normotensive rats was subjected to hypokalemia for 60 min unless fibrillation occurred earlier. Myocardial tissue taken during control, burst pacing and hypokalemia conditions were examined for ultrastructural and gap junction protein, connexin-43, alterations. The results showed that old guinea pig heart is prone, while young resistant to atrial fibrillation and that hypertensive rat heart is more vulnerable than normotensive rat heart to ventricular fibrillation. In correlation with these findings it was revealed that age- as well as hypertension-related myocardial remodeling is accompanied by decreased intercellular coupling and down-regulation of conexin-43. Further deterioration of cell-to-cell coupling was observed most likely due to burst pacing and hypokalemia induced calcium overload. We suggest that structural substrate for arrhythmogenesis includes impairment of intercellular junctions. Thus, age- and hypertension-related maladaptation of the heart may account for its increased susceptibility to cardiac fibrillation.

Effect of Estradiol on Rat Ileum

1. Sex hormones may influence gastrointestinal motility and thus may be responsible for symptoms that are common during pregnancy or hormone replacement therapy. The purpose of this study was to evaluate the effect of estradiol on the gut. 2. Segments of rat ileum (n=9) were suspended in an organ bath and exposed to increasing concentrations of carbachol, in the presence or absence of 17beta-estradiol. 17beta-estradiol markedly reduced the force developed by the ileum in response to carbachol. 3. These results suggest that estradiol reduces gastrointestinal motility.

Ventricular defibrillating properties of catecholamine uptake inhibitors.

Ventricular fibrillation (VF) is a fatal event in humans unless electrical defibrillation is applied within minutes. Recent publications describe spontaneous termination of VF in various animals and even in humans. Certain drugs can transfer a fatal, sustained VF (SVF) into a self-terminating, transient VF (TVF). Based on results obtained in animals of various species and ages, we have suggested that the occurrence of TVF requires a high cardiac catecholamine level at the time of VF. According to our hypothesis, drugs which decrease catecholamine reuptake by the sympathetic nerve terminals will increase the ability of the heart ventricles to defibrillate spontaneously. In the present study, we examined the effects of desipramine, maprotiline, mianserin, iprindole, cocaine and amphetamine on the type of VF in cats exhibiting SVF prior to the treatment. The results show that the ability of these compounds to transfer SVF to TVF is closely related to their potency to inhibit catecholamine reuptake. The establishment of the catecholamine related mechanisms of TVF may lead to the development of a new class of antiarrhythmic-defibrillatory drugs.

The effect of tricyclic antidepressants on ventricular fibrillation and collateral blood supply following acute coronary occlusion.

SummaryIn previous studies, we showed that dibenzepin HCl (D) and other tricyclic antidepressants (TCAD), given either before or during occlusion of the left anterior descending artery (LAD), decreased the incidence of ventricular fibrillation (VF) following occlusion and reperfusion. Moreover, once VF develops in treated animals, it changes into a transient type, reverting spontaneously to a sinus rhythm. In the treated cats, retrograde perfusion of the occluded coronary artery was observed, most likely as a result of increased collateral blood flow. This latter effect is the subject of the present study.The LAD was occluded at its origin in 43 cats, 28 of which were treated either with D or with 5-iminodibenzyl HCl; the remaining 15 were untreated controls. Two hours after the occlusion, methylene blue was injected into the left atrium to determine color demarcation between the perfused and unperfused myocardium, and the cat was then killed. After fixing for 2 or 3 days in 4% formaldehyde, the hearts were sectioned transversely. The results showed that in the 15 control cats, the blood-supplied (blue) area ranged between 16% and 56% of the left ventricular muscle (mean 39%), while in the 28 treated cats the blue area was between 44% and 83% (mean 66%). These results clearly indicate the beneficial effect of TCAD on the blood supply of the occluded area and can explain, in part, the ability of these drugs to prevent VF even if infused after the coronary occlusion, and their protective effect against VF following reperfusion. No other antiarrhythmic drugs have been shown to possess this latter action.On the basis of all our results, clinical trials to investigate the effect of TCAD seem to be warranted.

A SELF-PROTECTING SERVO-MODEL FOR EXPLANATION OF THE MECHANISM INVOLVED IN SPONTANEOUS VENTRICULAR DEFIBRILLATION

Ventricular fibrillation (VF) is the most life-threatening arrhythmia. It has been suggested that VF in humans is always sustained. Recent publications indicated that VF can be either sustained (SVF) or transient (TVF), reverting spontaneously into sinus rhythm. In previous studies we have hypothesized that TVF requires, during VF, a high cardiac catecholamine level ([CA]). Since during VF sympathetic activity is enhanced, the question arises of why VF is sustained in the majority of cases. Looking on the living body as a self-protecting servo-mechanism, we propose a servo-model that on the one hand describes the mechanism involved in TVF and on the other proposes a therapeutic procedure which can help the heart in its effort to transform VF into TVF. Our model has been examined by various experimental studies. The results obtained strongly support our hypothesis.

ETHMOZINE AND ETHACIZINE - NEW ANTIARRHYTHMIC DRUGS WITH DEFIBRILLATING PROPERTIES

Ventricular fibrillation (VF) is a life-threatening arrhythmia that leads to death unless electrical defibrillation is applied in time. Recent publications indicate that VF can be either sustained (SVF), requiring electrical defibrillation, or transient (TVF), reverting spontaneously into sinus rhythm. Since VF cannot be totally prevented by drugs, a new antiarrhythmic therapeutic approach has been proposed: drug-induced enhancement of the ability of the heart to defibrillate by itself. In this study we examined the defibrillating potency of two antiarrhythmic phenothiazines, ethmozine (ETM) and ethacizine (ETA), as well as their effects on catecholamine uptake and on the electrophysiological properties of the myocardial cell membrane. The antiarrhythmic-defibrillatory activity was examined in cats; the inhibitory effect on [3H]-norepinephrine (NE) uptake was examined in rat brain synaptosomes, and the electrophysiological membrane effects were examined by microelectrode recordings in perfused strips of heart ventricle from guinea-pigs. The results indicate that: 1. ETA exhibits similar but stronger antiarrhythmic-defibrillating and NE reuptake inhibitory effects than ETM; 2. ETA at 10-6 M decreases ventricular conduction time and increases Vmax while ETM at this concentration does not change them; 3. The defibrillating ability of the drugs can be related to their inhibitory potency on NE reuptake. We suggest that the risk of sympathomimetic arrhythmogenicity is prevented by the previously described, membrane stabilizing Class 1 antiarrhythmic properties of these drugs.