Snake alpha-neurotoxin binding site on the Egyptian cobra (Naja haje) nicotinic acetylcholine receptor Is conserved.

Evolutionary success requires that animal venoms are targeted against phylogenetically conserved molecular structures of fundamental physiological processes. Species producing venoms must be resistant to their action. Venoms of Elapidae snakes (e.g., cobras, kraits) contain alpha-neurotoxins, represented by alpha-bungarotoxin (alpha-BTX) targeted against the nicotinic acetylcholine receptor (nAChR) of the neuromuscular junction. The model which presumes that cobras (Naja spp., Elapidae) have lost their binding site for conspecific alpha-neurotoxins because of the unique amino acid substitutions in their nAChR polypeptide backbone per se is incompatible with the evolutionary theory that (1) the molecular motifs forming the alpha-neurotoxin target site on the nAChR are fundamental for receptor structure and/or function, and (2) the alpha-neurotoxin target site is conserved among Chordata lineages. To test the hypothesis that the alpha-neurotoxin binding site is conserved in Elapidae snakes and to identify the mechanism of resistance against conspecific alpha-neurotoxins, we cloned the ligand binding domain of the Egyptian cobra (Naja haje) nAChR alpha subunit. When expressed as part of a functional Naja/mouse chimeric nAChR in Xenopus oocytes, this domain confers resistance against alpha-BTX but does not alter responses induced by the natural ligand acetylcholine. Further mutational analysis of the Naja/mouse nAChR demonstrated that an N-glycosylation signal in the ligand binding domain that is unique to N. haje is responsible for alpha-BTX resistance. However, when the N-glycosylation signal is eliminated, the nAChR containing the N. haje sequence is inhibited by alpha-BTX with a potency that is comparable to that in mammals. We conclude that the binding site for conspecific alpha-neurotoxin in Elapidae snakes is conserved in the nAChR ligand binding domain polypeptide backbone per se. This conclusion supports the hypothesis that animal toxins are targeted against evolutionarily conserved molecular motifs. Such conservation also calls for a revision of the present model of the alpha-BTX binding site. The approach described here can be used to identify the mechanism of resistance against conspecific venoms in other species and to characterize toxin-receptor coevolution.

Paucity of CFTR current but modest CFTR immunoreactivity in non-diseased human ventricle.

We looked for evidence of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in non-diseased human ventricle. In rabbit and guinea pig the CFTR current is present in the highest density in subepicardial ventricular myocytes. In the present study, whole-cell patch-clamp was used to determine if a CFTR-like chloride current (I(CFTR,card)) can also be activated in human subepicardial ventricular myocytes. No evidence for I(CFTR,card) was detected in these electrophysiological studies when 10 microM forskolin was applied to 23 different cells from 4 donor hearts. Consistent with our previous results, a swelling-induced chloride current (I(Cl,swell)) could be observed after cell inflation. The enzymatic digestion of human ventricle to release single myocytes may have affected our ability to detect I(CFTR,card). Therefore, we looked for anti-CFTR immunoreactivity in slices of left ventricular free wall. A strong immunoreactivity signal was observed in guinea pig ventricle, a positive control. Background staining levels were seen in dog ventricle, a negative control tissue. Human anti-CFTR immunoreactivity was slightly above background. This low level of anti-CFTR immunoreactivity is consistent both with reports that CFTR mRNA is detectable in human ventricle and our inability to detect a significant I(CFTR,card) current density.

Cardiac swelling-induced chloride current is enhanced by endothelin.

Endothelins (ETs) are a family of peptide hormones that act on G protein-coupled ET(A) and ET(B) receptors. ETs exert inotropic and chronotropic actions in the heart. Myocardial ischemia is associated with increased plasma levels of ET and cell swelling. We examined the effect of ETs on dog atrial swelling-induced chloride current (I(Cl,swell)). Whole-cell patch clamp was used; 10 nM ET-1 or ET-2 increased I(Cl,swell) by approximately twofold. ET-2 had no effect if I(Cl,swell) activation was prevented by hypertonic superfusate. Outward ET-2-induced current was blocked by 150 microM DIDS more effectively than inward current. Overnight pretreatment with phorbol 12-myristate 13-acetate (1.6 microM), pertussis toxin (100 ng/ml), or dialysis of the cell with 300 microM 2'-deoxyadenosine 3'-monophosphate, a P-site inhibitor of adenylyl cyclase, did not diminish the effect of ET-2. The effect of ET-2 was blocked by an ET(A1)- (BQ123), but not an ET(B)-selective (BQ788) antagonist. ET-2-induced currents were inhibited approximately 70% by PD 98059 (30 microM), a selective MAPK kinase (MEK) blocker. PD 98059 did not affect basal whole cell current or I(Cl,swell) before exposure to ET-2. The data suggest that MEK activity is not required for activation of atrial I(Cl,swell) but that ET-2 stimulates I(Cl,swell) by a MEK-dependent pathway.

Insights into the structure, distribution and function of the cardiac chloride channels.

This review describes the properties and distribution of the three major types of chloride currents that have been studied in cardiac tissue. These include a cAMP- and protein kinase A-dependent current, a calcium-activated current and a swelling-induced current. The study of cardiac anion currents is a less mature field than the study of cardiac cation currents. Consequently, less is known regarding the structure, molecular identity and physiological role of anion currents in comparison to cardiac cation currents. Where known, the available molecular and structural information is also discussed. Although there is no proven physiological role for cardiac chloride currents, the possible clinical electrophysiological roles of cardiac chloride currents are discussed.

Protein kinase C stimulates swelling-induced chloride current in canine atrial cells.

The whole-cell patch-clamp technique was used to study the effect of protein kinase C (PKC) stimulation and alpha-adrenergic agonists on the swelling-induced chloride current (ICl,swell) in canine atrial cells. ICl,swell was activated by positive-pressure inflation. 4beta-Phorbol 12, 13-dibutyrate (PDBu) concentration-dependently stimulated ICl,swell. PDBu (500 nM) increased the current density of ICl,swell from 9.1+/-1.3 to 24.2+/-4.8 pA/pF at +20 mV (n=4). This effect developed slowly, reaching a steady-state after more than 5 min of exposure. 4alpha-Phorbol 12, 13-dibutyrate (4alpha-PDBu, 500 nM), an inactive analogue of PDBu, did not affect ICl,swell. The effect of PDBu was inhibited by bisindolylmaleimide I. After down regulation of PKC by phorbol 12-myristate 13-acetate (PMA, 1.6 microM, 24 h), ICl,swell no longer responded to PDBu (n=4). Neither the basal whole-cell current (prior to cell inflation) nor inflation-induced ICl,swell were affected by PKC down regulation. Phenylephrine did not affect ICl,swell. We conclude that PKC activity stimulates and does not prevent the activation of dog atrial ICl,swell. These results contrast with reports of PKC-dependent inhibition of rabbit atrial ICl,swell and currents conducted by ClC-3, a putative clone for ICl,swell. The data suggest species-dependent variations in the modulation of cardiac ICl,swell by PKC.

Isoprenaline can activate the acetylcholine-induced K+ current in canine atrial myocytes via Gs-derived betagamma subunits.

1. G protein betagamma subunits activate the acetylcholine-induced potassium current IK,ACh. There is no evidence of specificity at the level of the betagamma subunits. Therefore all G protein-coupled receptors in atrial myocytes should be able to activate IK,ACh. Paradoxically, it is often stated that isoprenaline does not activate IK,ACh. Rationales to explain this negative result include insufficient concentrations of Gs in the atrium or restricted access of Gs-derived betagamma subunits to the IK,ACh channel. We took advantage of a non-specific increase in Gs that results after infection with adenovirus. 2. Adenoviral infection unmasked a 1 microM isoprenaline-induced IK,ACh which was prevented by propranolol. Isoprenaline occasionally activated IK,ACh in uninfected and freshly dissociated atrial myocytes but the effect was larger and more consistent in infected myocytes. 3. Pertussis toxin pretreatment (100 ng ml-1 overnight) did not block the effect of isoprenaline. The effect of isoprenaline became persistent if cells were pretreated with cholera toxin (200 ng nl-1). 4. Signal transduction events distal to adenylyl cyclase were not involved in isoprenaline-induced IK,ACh. Forskolin (10 microM) did not activate IK,ACh. Inhibition of adenylyl cyclase with cytoplasmic application of 300 microM 2'-deoxyadenosine 3'-monophosphate did not prevent the activation of IK,ACh by isoprenaline. 5. Cytoplasmic application of a betagamma binding peptide derived from the C terminus of beta-adrenergic receptor kinase 1 (50 microM) prevented the effect of isoprenaline on IK,ACh. The peptide did not prevent the stimulation of the L-type calcium current by isoprenaline. 6. The results indicate that beta-adrenoceptors can activate IK,ACh in atrial myocytes through the release of betagamma subunits from Gs.

Delayed activation of cardiac swelling-induced chloride current after step changes in cell size.

INTRODUCTION: A lag phase has been reported for the activation of cardiac swelling-induced chloride currents. Prior demonstrations of this lag used methods that produce gradual changes in cell size, making interpretation and quantification of the time course problematic. METHODS AND RESULTS: Isolated dog atrial cells were studied using the whole cell, patch clamp technique. Step changes in cell size were produced by application of transient pulses of positive pressure, and the time course for activation of the swelling-induced chloride current was observed. There was a distinct temporal dissociation between size changes and current activation that was temperature sensitive. Activation half-times were 98 +/- 31 seconds and 586 +/- 112 seconds at 36 degrees C and room temperature, respectively. Swelling-induced chloride currents were evoked in a higher percentage of cells at 36 degrees C (83%) compared with room temperature (50%). CONCLUSION: Cardiac swelling-induced chloride current activates with a distinct lag after step changes in cell size. The activation time course is temperature sensitive. These observations are consistent with the notion that signal transduction events, and not simply membrane stretch, are required for the activation of cardiac swelling-induced chloride current.

ATP-dependent activation of the atrial acetylcholine-induced K+ channel does not require nucleoside diphosphate kinase activity.

Prior reports by others have shown that cytoplasmically applied ATP can activate the acetylcholine-induced K+ channel in inside-out atrial membrane patches when no guanine nucleotides are present in the solution bathing the cytosolic face of the membrane. A nucleoside diphosphate kinase mechanism was proposed to explain the activation by ATP. We show in the present study that cytoplasmic adenylylimidodiphosphate mimics the activation by ATP. Unlike ATP, the activation by adenylylimidodiphosphate does not subside on washout. Although commercially available adenylylimidodiphosphate is contaminated by guanylylimidodiphosphate, the activation by adenylylimidodiphosphate still occurs after HPLC purification to remove guanine nucleotide contamination. Adenylylimidodiphosphate does not support phosphotransferase activity by nucleoside diphosphate kinase. Therefore, nucleoside diphosphate kinase activity cannot explain the activation of atrial acetylcholine-induced K+ current by ATP and adenylylimidodiphosphate. We hypothesize that the activation by millimolar concentrations of ATP is due to binding of adenine nucleotide to the guanine nucleotide binding site of the G protein(s) responsible for stimulating the acetylcholine-induced K+ current.

Modulation of dog atrial swelling-induced chloride current by cAMP: protein kinase A-dependent and -independent pathways.

1. The modulation of dog atrial swelling-induced chloride current (I(Cl,swelling)) by cAMP-elevating agents was studied. Forskolin (10 microM) or isoprenaline (1 microM) exerted multiple effects. Although the pattern between cells was variable, there was, in general, a stimulatory action and a more slowly developing inhibitory effect. 2. In any given cell, the response to forskolin or isoprenaline was qualitatively similar suggesting that all of the responses were dependent on stimulation of adenylyl cyclase. The effects of forskolin or isoprenaline on I(Cl,swelling) were inhibited by intracellular dialysis with a P-site inhibitor of adenylyl cyclase, 2'-deoxyadenosine 3'-monophosphate (300 microM). 3. Intracellular dialysis with a peptide inhibitor of protein kinase A (PKI(6-22); 100 microM) blocked the inhibitory response to forskolin or isoprenaline and all cells responded with a monophasic stimulation of I(Cl,swelling). 4. After intracellular dialysis of cells with PKI(6-22) (100 microM) and cAMP (100 microM), current amplitude was not further stimulated by forskolin. 5. After intracellular dialysis with PKI(6-22) and adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), forskolin stimulated I(Cl,swelling) and the effect of forskolin subsided after it was washed out. 6. In conclusion, there are dual pathways by which cAMP can modulate dog atrial cell I(Cl,swelling). Inhibition results from protein kinase A (PKA)-dependent phosphorylation. In addition, a stimulatory pathway exists that is independent of phosphorylation by PKA or other cellular kinases. Although alternative explanations are possible, the stimulatory effect of cAMP may represent a direct modulation of I(Cl,swelling).

Cardiac swelling-induced chloride current depolarizes canine atrial myocytes.

The effect of the cardiac swelling-induced chloride current (I(Cl,swell)) on the transmembrane potential was examined. Osmotic swelling affected the resting potassium current through an apparent dilution of intracellular potassium. Inflating cells by applying positive pressure to the patch electrode prevented the effect on the resting potassium current. Inflation depolarized dog atrial myocytes when the recording electrodes contained either 17 or 42 mM Cl-. The depolarization coincided with activation of I(Cl,swell) and was antagonized by the chloride-channel blocker niflumic acid. Substituting extracellular chloride with the more permeant ion SCN- shifted the reversal potential for I(Cl,swell) to more negative values and antagonized inflation-induced depolarization. The depolarization was accentuated by replacing extracellular chloride with a less permeant ion, aspartate. We conclude that activation of I(Cl,swell) in atrial cells causes significant depolarization of the resting membrane. The outward rectification of I(Cl,swell) and the high cell membrane resistance during the action potential plateau suggest that I(Cl,swell) will also have significant effects on atrial action potential configuration.

Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current.

The effect of tyrosine protein kinase inhibitors on the swelling-induced chloride current (ICl-swelling) of dog atrial myocytes was studied using the whole-cell patch-clamp recording technique. Currents were measured during hyperpolarizing voltage ramps with potassium currents blocked by cesium. Osmolarity was varied using mannitol. Exposure to hypotonic solution (approximately 249 mosmol/kg) activated ICl-swelling. Hypertonic solution (approximately 363 mosmol/kg) was used to shrink swollen cells and turn off ICl-swelling. In studies on the acute effect of tyrosine protein kinase inhibitors each cell was swollen three separate times. Control, treatment, and washout ICl-swelling were compared. Genistein (50-80 microM) prevented reactivation of ICl-swelling without affecting cell size. The effect of genistein partially subsided upon washout. The effect of genistein on ICl-swelling was not mimicked by 80 microM daidzein, a related compound that does not inhibit tyrosine protein kinases. When intracellular adenosine 5'-O-(3-thiotriphosphate (ATP[gamma S]) was used, genistein did not prevent the reactivation of ICl-swelling. Intracellular ATP[gamma S] did not result in a persistent activation of ICl-swelling when cell size was returned to control. Acute exposure to 1 microM herbimycin A or 100 microM tyrphostin 51 did not prohibit the activation of ICl-swelling. A 24-h exposure to 1 microM herbimycin A did inhibit ICl-swelling. The results provide important clues regarding the activation mechanism for ICl-swelling and suggest that a tyrosine protein phosphorylation may be necessary, but not sufficient, for activation of ICl-swelling.

Forskolin stimulates swelling-induced chloride current, not cardiac cystic fibrosis transmembrane-conductance regulator current, in human cardiac myocytes.

Whole-cell patch clamp was used to look for cystic fibrosis transmembrane-conductance regulator (CFTR)-like chloride currents in calcium-tolerant human cardiac myocytes. Potassium-containing solutions were used initially. Steady state currents were measured with hyperpolarizing ramps (-16.25 mV/s). Peak net inward currents during voltage steps from -50 to +5 mV were used as an index of L-type calcium current. Isoproterenol (1 mumol/L) or forskolin (10 mumol/L) were used in attempts to evoke CFTR-like chloride current. No forskolin- or isoproterenol-induced steady state current was found in any of 17 atrial cells from seven patients in the absence of cell swelling. Every cell exhibited a large increase in net inward current in response to forskolin, suggesting that cAMP-dependent stimulation of L-type calcium current was present. Swelling with osmotic stress induced an outwardly rectifying steady state current with a reversal potential close to the chloride equilibrium potential. Once this current was activated, exposure to forskolin caused a further increase that subsided on washout (four of four cells, two patients). The atrial swelling-induced current was studied in more detail by using cesium-containing solutions. The current was determined to be a chloride current because the reversal potential was sensitive to changes in intracellular chloride and outward currents were blocked by 150 mumol/L DIDS. Ventricular cells were isolated from five failing human hearts. No CFTR-like current was found in any of 17 cells. In eight of eight ventricular cells, a swelling-induced current was found. The amplitude of the swelling-induced current was enhanced by forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacologic properties of the swelling-induced chloride current of dog atrial myocytes.

INTRODUCTION: Swelling-induced chloride currents may contribute to cardiac electrical activity and cell volume regulation. Identification of selective blockers would aid in understanding the functional contribution(s) of this current. METHODS AND RESULTS: Dog atrial cells were used to investigate the pharmacologic properties of the swelling-induced chloride current. Whole cell patch clamp was used. Swelling-induced chloride current was activated by osmotic stress. Initially, the chloride selectivity and calcium independence of the swelling-induced current in dog atrial cells was demonstrated. Subsequently, a number of putative chloride channel blockers were examined. Anthracene-9-carboxylic acid (1 mM) and dideoxyforskolin (100 microM) and extracellular cAMP (5 mM) were found to partially inhibit the swelling-induced chloride current (approximately 50%, 80%, and 10% inhibition, respectively). Niflumic acid (100 microM), nitrophenylpropylamino benzoate (NPPB; 10 to 40 microM), and (+) 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxy-1H-inden -5-yl)oxy d acetic acid (indanyloxyacetic acid; IAA-94; 100 microM) could fully inhibit the swelling-induced chloride current without decreasing cell size. DIDS (100 microM) and dinitrostilbene disulfonic acid (DNDS; 5 mM) fully inhibited outward currents but only partially inhibited inward current. CONCLUSIONS: Niflumic acid, IAA-94, and NPPB were identified as full blockers of cardiac swelling-induced chloride current. Nonspecific effects were identified for each of the full blockers. Experiments that use these agents as functional antagonists should be carefully designed and interpreted with caution.

Downregulation of adenylylcyclase types V and VI mRNA levels in pacing-induced heart failure in dogs.

We have shown that the heart expresses two distinct forms of adenylylcyclase mRNA, types V and VI. In this study we have characterized the expression of these two mRNA species in heart failure generated by overdrive pacing at a rate of 240 beats/min. After 4 wk, left ventricular end-diastolic pressure and heart rate increased significantly with the appearance of signs of heart failure, i.e., edema, ascites, and exercise intolerance. Basal as well as forskolin-stimulated adenylylcyclase activities decreased significantly, which was accompanied by a reduction in the steady state mRNA levels of adenylylcyclase types V and VI. These data suggest that in this model of cardiomyopathy, the downregulation of adenylylcyclase catalytic activity results, at least in part, from a reduction in the steady state levels of types V and VI adenylylcyclase mRNA levels.

Spontaneous activity in transgenic mouse heart: comparison of primary atrial tumor with cultured AT-1 atrial myocytes.

INTRODUCTION: We have generated transgenic animals that heritably develop atrial tumors composed of differentiated proliferating cardiomyocytes. Experiments were initiated to characterize the electrical properties of these cells. METHODS AND RESULTS: We show that the primary atrial tumors are composed of discrete foci that exhibit spontaneous automatic activity. A direct correlation was observed between tumor size and firing rate of these foci. In addition to the primary atrial tumors, we examined the properties of cultured cardiomyocytes isolated from a transplantable transgenic tumor lineage (designated AT-1 cells). Cultured AT-1 cells are also spontaneously automatic. The action potential configuration from these preparations is similar to that observed in nontransgenic atrial cardiomyocytes, albeit somewhat more depolarized and of longer duration. As would be expected for cardiomyocytes of atrial origin, the transgenic cardiomyocyte preparations hyperpolarize during muscarinic stimulation due to increased K+ conductance mediated by a pertussis toxin sensitive G-protein. Assessment of pharmacologic blockage of the "if" pacemaker current suggests that the automaticity of both transgenic cardiomyocyte preparations may be of novel origin. In this context, the cultured AT-1 cells showed spontaneous behavior that was clearly of cellular origin; this activity was manifest as transient bursts of electrical activity followed by periods of electrical quiescence. This bursting pattern is unusual for normal adult cardiomyocytes, but has been observed in several other cell types. In the primary tumors, automatic behavior may arise from a similar cellular origin or alternatively from a microreentrant phenomena. CONCLUSION: Primary tumors and AT-1 cells show essential atrial electrophysiology with important novel features.

Swelling-induced chloride-sensitive current in canine atrial cells revealed by whole-cell patch-clamp method.

An isoproterenol-induced chloride current has been detected in ventricular myocytes from guinea pig and rabbit but has not been found in canine ventricular cells. This investigation was undertaken to determine whether canine atrial cells possessed such a current. Steady-state currents were examined with potassium currents blocked by cesium. In whole-cell patch-clamp experiments, an isoproterenol-induced chloride current could not be detected shortly after patch rupture. However, whole-cell current in the absence of isoproterenol increased over time after patch rupture. The spontaneously activating steady-state current was outwardly rectifying with a reversal potential of approximately -25 mV. The current that developed over time was sensitive to variation in extracellular chloride concentration and was partially blocked by anthracene-9-carboxylic acid. Isoproterenol could enhance the amplitude of this current once it developed. Although isosmotic pipette filling and extracellular solutions were used, cell swelling was found to be the cause of the increase in whole-cell conductance that was observed during whole-cell patch-clamp experiments. The development of the current and the associated cell swelling could be prevented with the addition of 50-75 mM mannitol to the extracellular solution. The current could be observed in perforated patch recordings with nystatin when extracellular osmolarity was low (221 mosm/kg) but not when the extracellular solution was isosmotic (293 mosm/kg). Cardiac chloride currents have the potential to depolarize the resting membrane potential and cause abnormal automaticity. Chloride currents can also decrease the refractory period through a reduction in action potential duration.(ABSTRACT TRUNCATED AT 250 WORDS)

The isoproterenol-induced chloride current and cardiac resting potential.

Isoproterenol can induce potentially arrhymthogenic depolarizations of the resting membrane of single guinea-pig ventricular myocytes. This effect on resting potential of single guinea-pig ventricular myocytes differs qualitatively from results obtained by others from intact cardiac muscle preparations and also differs from our experience with single dog ventricular myocytes. We performed experiments on dog and guinea-pig ventricular myocytes in an attempt to clarify the effects of isoproterenol on the resting potential of mammalian ventricular myocytes. Voltage recordings with 40-60 M omega 3 M potassium chloride filled microelectrodes revealed an isoproterenol-induced depolarization of 4.3 +/- 1.0 mV in guinea-pig but no depolarization in dog myocytes. Activation of an outwardly rectifying chloride current is responsible for the isoproterenol-induced depolarization of guinea-pig ventricular myocytes. Our whole cell patch clamp recordings consistently revealed such a current in guinea-pig cells but always failed to demonstrate an isoproterenol-induced chloride current in dog myocytes under identical conditions. In contrast to single cells, isoproterenol did not depolarize intact guinea-pig papillary muscle when potential was recorded with 40-60 M omega 3 M KCl filled electrodes. Furthermore, we saw no depolarization in single guinea-pig myocytes when recording electrodes did not contain chloride. We conclude that: (1) despite activation of a chloride current, isoproterenol does not significantly depolarize guinea-pig ventricular muscle unless the driving force for the current at the resting potential is increased by elevating [Cl]i above physiological levels, and (2) an isoproterenol-induced chloride current, although demonstrable in guinea-pig ventricular cells, is not present in healthy dog ventricular cells.

Antiarrhythmic actions of the ATP-regulated K+ current activated by pinacidil.

We tested the hypothesis that a selective increase in membrane current, as contrasted with the decreases in currents caused by most antiarrhythmic agents, would be an effective antiarrhythmic intervention. We studied models of early afterdepolarizations (EADs), delayed afterdepolarizations (DADs), and abnormal automaticity in single canine ventricular myocytes using intracellular microelectrodes or patch electrodes. EADs were induced by injected current, Bay K 8644 (0.5-1 microM), or ketanserin (1.0 microM); DADs were induced by ouabain intoxication (2 x 10(-7) M); and abnormal automaticity was induced by exposure to barium (0.25 mM). To increase outward K+ current, we used pinacidil and the protein kinase C activator 4 beta-phorbol 12,13-dibutyrate (PDBu). Under control conditions, 10-100 microM pinacidil caused a concentration-dependent and reversible decrease in action potential duration and an increase in steady-state outward current; both effects were blocked by glibenclamide and thus presumably reflected changes in the ATP-regulated potassium current. Pinacidil increased the current required to induce EADs and abolished EADs caused by Bay K 8644 or ketanserin. After exposure of myocytes to ouabain, pinacidil caused a decrease in action potential duration and diminished or abolished DADs. Finally, pinacidil arrested abnormal automaticity caused by Ba2+. PDBu (30 nM) shortened action potential duration without altering plateau amplitude in some of the myocytes. In these cells the depolarizing current needed to produce an EAD was increased by over 70%; outward potassium current tails were also increased, an effect consistent with an increase of the repolarizing potassium current (IK). These findings show that each of the mechanisms for abnormal impulse generation can be effectively antagonized by an increase in outward current and suggest to us that selective augmentation of a repolarizing current, possibly IK, might be a reasonable antiarrhythmic intervention.

Role of G proteins in the acetylcholine-induced potassium current of canine atrial cells.

The acetylcholine-induced opening of potassium channels depends on GTP-binding proteins in the chick, guinea pig, frog, and rat. In contrast, Bubien and Woods (Biochem. Biophys. Res. Commun. 142: 1039-1045, 1987) have recently postulated that the acetylcholine response in cultured canine atrial cells may be independent of GTP-binding proteins. In whole cell patch-clamp experiments using cultured canine atrial cells, we did not detect an effect of GTP (10(-4) M) in the pipette solution on the acetylcholine-induced potassium current. However, 500 microM guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) in the pipette diminished the response to acetylcholine. Pertussis toxin (30 ng/ml for 24 h) blocked the response to acetylcholine. With guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 3 microM) in the patch pipette, acetylcholine irreversibly increased membrane conductance. The current-voltage relationship for the persistently activated current was similar to that induced by acetylcholine. We conclude that the acetylcholine-induced potassium current in canine atrial cells behaves like that seen in other species and depends on GTP-binding proteins.

Positive vs. negative inotropic effects of carbachol in avian atrial muscle: role of Ni-like protein.

Treatment with pertussis toxin not only prevents inhibitory effects (e.g., reduced adenylate cyclase activity, decreased voltage-dependent Ca2+ entry, increased K+ efflux, and negative inotropy) but also unmasks stimulant effects (e.g., membrane depolarization and positive inotropy) of carbachol in chick atria. Pertussis toxin prevents transducer proteins (Ni and No) from linking muscarinic receptors to either adenylate cyclase and Ca2+ channels or K+ channels. However, pertussis toxin treatment does not block N proteins linking the muscarinic receptor to stimulant membrane effects. Membrane depolarization by carbachol, attributed by others to increased Na+ entry, may stimulate Na-Ca exchange and positive inotropy, perhaps by activation of phospholipase D. Alternatively, carbachol could increase inositol triphosphate content and thereby release Ca2+ from the sarcoplasmic reticulum to increase the force of contraction. The ability of carbachol to increase phosphoinositide hydrolysis is resistant to pertussis toxin. The second-messenger role of phospholipid metabolites provides a foundation for testing the hypothesis that such metabolites are eventually involved in the stimulant actions of carbachol seen in pertussis toxin-treated preparations.