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.

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.

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)

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.

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.

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.

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.

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).

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.

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.