1. Outward currents were studied in myocytes isolated from human atrial and subepicardial ventricular myocardium using the whole-cell voltage clamp technique at 220C. The Nae current was inactivated with prepulses to -40 mV and the Ca2P current was eliminated by both reducing extracellular [Ca2+] to 0 5 mm and addition of 100 /M CdCl2 to the bath solution. 2. In human myocytes, three different outward currents were observed. A slowly inactivating sustained outward current, I,,, was found in atrial but not ventricular myocytes. A rapidly inactivating outward current, It., of similar current density was observed in cells from the two tissues. An additional uncharacterized non-inactivating background current of similar size was observed in atrial and in ventricular myocytes.3. It. and I4o could be differentiated in atrial myocytes by their different kinetics and potential dependence of inactivation, and their different sensitivities to block by 4-aminopyridine, suggesting that two individual channel types were involved.4. In atrial cells, inactivation of I. was more rapid and steady-state inactivation occurred at more negative membrane potentials than in ventricular cells. Furthermore, the recovery of It. from inactivation was slower and without overshoot in atrial myocytes. In addition, 4-aminopyridine-induced block of It. was more efficient in atrial than in ventricular cells. These observations suggest that the channels responsible for atrial and ventricular It. were not identical. 5. We conclude that the differences in outward currents substantially contribute to the particular shapes of human atrial and ventricular action potentials. The existence of I.. in atrial cells only provides a clinically interesting target for anti-arrhythmic drug action, since blockers of Iko would selectively prolong the atrial refractory period, leaving ventricular refractoriness unaltered.The action potential shape shows characteristic differences between atrium and ventricle in several animal models, e.g. the guinea-pig (Hume & Uehara, 1985). Inhomogeneities in repolarizing currents between atria and ventricles generated these action potential shape differences. The action potential of human atrium is markedly shorter than that of ventricle, and possesses no clear plateau phase (Trautwein, Kassebaum, Nelson & Hecht, 1962). A careful comparison between the outward currents of human atrial and ventricular myocytes has, however, not been undertaken.The transient outward current (It.) is a major repolarizing current in both human atrium (Escande, Coulombe, Faivre,

Vascular endothelium appears to be a unique organ. It not only responds to numerous hormonal and chemical signals but also senses changes in physical parameters such as shear stress, producing mediators that modulate the responses of numerous cells, including vascular smooth muscle, platelets, and leukocytes. In many cases, the initial response of endothelial cells to these diverse signals involves elevation of cytosolic Ca 2+ and activation of Ca 2+-dependent enzymes, including nitric oxide synthase and phospholipase A 2 . Both the release of Ca 2+ from intracellular stores, most likely the endoplasmic reticulum, and the influx of Ca 2+ from the extracellular space contribute to the [Ca 2+ ]| increase. The most important trigger for Ca 2+ release is inositol 1,4,5 -trisphosphate, which is generated by the action of phospholipase C, a plasmalemmal enzyme activated in many cases by the receptor-G protein cascade. Ca 2+ influx appears to be related to the activity of receptor-G protein-enzyme complex and to the degree of fullness of the endoplasmic reticulum but does not involve voltage-gated Ca 2+ channels. The magnitude of the Ca 2+ influx depends on the electrochemical gradient, which is modulated by the membrane potential, V m . Under basal conditions, V m is dominated by a large inward rectifier K + current. Some stimuli, e.g., acetylcholine, have been shown to hyperpolarize V m , thus increasing the electrochemical gradient for Ca 2+ , which appears to be modulated by activation of Ca 2+ -dependent K + and CI" currents. However, the lack of potent and specific blockers for many of the described or postulated channels (e.g., nonselective cation channel, Ca 2+ -activated Cl~ channel) makes an estimation of their effect on endothelial cell function rather difficult. involved in the contraction of endothelial cells and the increased permeability of microvessels in response to inflammatory agents (for review, see Reference 19). Thus, a detailed knowledge of intracellular Ca 2+ homeostasis is essential for our understanding of the physiology, pathophysiology, and pharmacology of endothelial cells. The following sections review the importance of the endothelium in various disease states and the mechanisms underlying cytoplasmic Ca 2+ regulation under basal and stimulated conditions. Importance of the Endothelium in VariousDisease States Increased systemic vascular tone, which is thought to result from enhanced circulating hormones (e.g., norepinephrine, angiotensin II), is among the most common hemodynamic findings in patients with heart failure. 20 However, heightened vasoconstriction correlates poorly with the plasma levels of these substances. 20 The poor correlation could possibly be explained by the involvement of local, endothelium-dependent factors, such as an imbalance of endothelium-derived relaxing and contracting factors.21 " 23 This view is supported by numerous studies conducted in isolated vascular segments as well as in intact laboratory animals and human subjects in a variety of disease states, i...

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In rat ventricle, two Ca2+-insensitive components of K+ current have been distinguished kinetically and pharmacologically, the transient, 4-aminopyridine (4-AP)-sensitive I to and the sustained, tetraethylammonium (TEA)-sensitive I K. However, a much greater diversity of depolarization-activated K+ channels has been reported on the level of mRNA and protein. In the search for electrophysiological evidence of further current components, the whole cell voltage-clamp technique was used to analyze steady-state inactivation of outward currents by conditioning potentials in a wide voltage range. Peak ( I peak) and late ( I late) currents during the test pulse were analyzed by Boltzmann curve fitting, producing three fractions each. Fractions a and b had different potentials of half-maximum inactivation ( V 0.5); the third residual fraction, r, did not inactivate. Fractions a for I peak and I late had similar relative amplitudes and V 0.5 values, whereas size and V 0.5 of fractions b differed significantly between I peak and I late. Only b of I peak was transient, suggesting a relation with I to, whereas a, b, and r of I late appeared to be three different sustained currents. Therefore, four individual outward current components were distinguished: I to( b of I peak), I K( a), the steady-state current I ss( r), and the novel current I Kx( b of I late). This was further supported by differential sensitivity to TEA, 4-AP, clofilium, quinidine, dendrotoxin, heteropodatoxin, and hanatoxin. With the exception of I to, none of the currents exhibited a marked transmural gradient. Availability of I K was low at resting potential; nevertheless, I K contributed to action potential shortening in hyperpolarized subendocardial myocytes. In conclusion, on the basis of electrophysiological and pharmacological evidence, at least four components contribute to outward current in rat ventricular myocytes.

Background-Clinical and experimental evidence suggest that the parasympathetic nervous system is involved in the pathogenesis of atrial fibrillation (AF). However, it is unclear whether changes in G-protein-coupled inward rectifying K ϩ current (I K,ACh ) contribute to chronic AF. Methods and Results-In the present study, we used electrophysiological recordings and competitive reverse-transcription polymerase chain reaction to study changes in I K,ACh and the level of the I K,ACh GIRK4 subunit in isolated human atrial myocytes and the atrial tissue of 39 patients with sinus rhythm and 24 patients with chronic AF. The density of I K,ACh was Ϸ50% smaller in myocytes from patients with AF compared with those in sinus rhythm, and this was accompanied by decreased levels of GIRK4 mRNA. The current density of the inward rectifying K ϩ current (I K1 ) was 2-fold larger during AF than in sinus rhythm, in correspondence with an increase in Kir2.1 mRNA. The larger I K1 in AF is consistent with more negative membrane potentials in right atrial trabeculae from AF patients. Moreover, action potential duration was reduced in AF, and the action potential shortening produced by muscarinic receptor stimulation was attenuated, indicating that the changes of I K1 and I K,ACh were functionally relevant. Conclusions-Chronic human AF induces transcriptionally mediated upregulation of I K1 but downregulation of I K,ACh and attenuates the muscarinic receptor-mediated shortening of atrial action potentials. This suggests that atrial myocytes adapt to a chronically high rate by downregulating I K,ACh to counteract the shortening of the atrial effective refractory period due to electrical remodeling.

Defining an appropriate and efficient assessment of drug‐induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc‐prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14‐based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B‐based “double‐negative” nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high‐dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double‐negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

We found an association between the Gbeta(3) 825T allele and amplitude of human atrial I(K1) and I(K,ACh). Increased background current density in TT carriers could shorten action potential duration and may be due to I(K,ACh) being constitutively active in this genotype.

Sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) have been reported to activate muscarinic receptor-activated inward rectifier K(+) current (I(K.ACh)) in cultured guinea pig atrial myocytes with similar nanomolar potency. Members of the endothelial differentiation gene (Edg) receptor family were recently identified as receptors for SPP; however, these receptors respond only to micromolar concentrations of SPPC. Here we investigated the sphingolipid-induced activation of I(K.ACh) in freshly isolated guinea pig, mouse, and human atrial myocytes. SPP activated I(K.ACh) in atrial myocytes from all three species with a similar nanomolar potency (EC(50) values: 4-8 nM). At these low concentrations, SPPC also activated I(K.ACh) in guinea pig myocytes. In contrast, SPPC was almost ineffective in mouse and human myocytes, thus resembling the pharmacology of the Edg receptors. Transcripts of Edg-1, Edg-3, and Edg-5 were detected in human atrial cells. Moreover, activation of I(K.ACh) by SPP was blocked by the Edg-3-selective antagonist suramin, which did not affect basal or carbachol-stimulated K(+) currents. In conclusion, these data indicate that I(K.ACh) activation by SPP and SPPC exhibits large species differences. Furthermore, they suggest that SPP-induced I(K.ACh) activation in human atrial myocytes is mediated by the Edg-3 subtype of SPP receptors.