Changes in intracellular calcium concentration influence beat-to-beat variability of action potential duration in canine ventricular myocytes.

The aim of the present work was to study the influence of changes in intracellular calcium concentration ([Ca(2+)]i) on beat-to-beat variability (short term variability, SV) of action potential duration (APD) in isolated canine ventricular cardiomyocytes. Series of action potentials were recorded from enzymatically isolated canine ventricular cells using conventional microelectrode technique. Drug effects on SV were evaluated as relative SV changes determined by plotting the drug-induced changes in SV against corresponding changes in APD and comparing these data to the exponential SV-APD function obtained with inward and outward current injections. Exposure of myocytes to the Ca(2+) chelator BAPTA-AM (5 µM) decreased, while Ca(2+) ionophore A23187 (1 µM) increased the magnitude of relative SV. Both effects were primarily due to the concomitant changes in APD. Relative SV was reduced by BAPTA-AM under various experimental conditions including pretreatment with veratridine, BAY K8644, dofetilide or E-4031. Contribution of transient changes of [Ca(2+)]i due to Ca(2+) released from the sarcoplasmic reticulum (SR) was studied using 10 µM ryanodine and 1 µM cyclopiazonic acid: relative SV was reduced by both agents. Inhibition of the Na(+)-Ca(2+) exchanger by 1 µM SEA0400 increased relative SV. It is concluded that elevation of [Ca(2+)]i increases relative SV significantly. More importantly, Ca(2+) released from the SR is an important component of this effect.

Role of action potential configuration and the contribution of C²âºa and K⁺ currents to isoprenaline-induced changes in canine ventricular cells.

BACKGROUND AND PURPOSE: Although isoprenaline (ISO) is known to activate several ion currents in mammalian myocardium, little is known about the role of action potential morphology in the ISO-induced changes in ion currents. Therefore, the effects of ISO on action potential configuration, L-type Ca²âº current (I(Ca)), slow delayed rectifier K⁺ current (I(Ks)) and fast delayed rectifier K⁺ current (I(Kr)) were studied and compared in a frequency-dependent manner using canine isolated ventricular myocytes from various transmural locations. EXPERIMENTAL APPROACH: Action potentials were recorded with conventional sharp microelectrodes; ion currents were measured using conventional and action potential voltage clamp techniques. KEY RESULTS: In myocytes displaying a spike-and-dome action potential configuration (epicardial and midmyocardial cells), ISO caused reversible shortening of action potentials accompanied by elevation of the plateau. ISO-induced action potential shortening was absent in endocardial cells and in myocytes pretreated with 4-aminopyridine. Application of the I(Kr) blocker E-4031 failed to modify the ISO effect, while action potentials were lengthened by ISO in the presence of the I(Ks) blocker HMR-1556. Both action potential shortening and elevation of the plateau were prevented by pretreatment with the I(Ca) blocker nisoldipine. Action potential voltage clamp experiments revealed a prominent slowly inactivating I(Ca) followed by a rise in I(Ks) , both currents increased with increasing the cycle length. CONCLUSIONS AND IMPLICATIONS: The effect of ISO in canine ventricular cells depends critically on action potential configuration, and the ISO-induced activation of I(Ks) - but not I(Kr) - may be responsible for the observed shortening of action potentials.

Interaction between Ca(2+) channel blockers and isoproterenol on L-type Ca(2+) current in canine ventricular cardiomyocytes.

AIM: The aim of this work was to study antagonistic interactions between the effects of various types of Ca(2+) channel blockers and isoproterenol on the amplitude of L-type Ca(2+) current in canine ventricular cells. METHODS: Whole-cell version of the patch clamp technique was used to study the effect of isoproterenol on Ca(2+) current in the absence and presence of Ca(2+) channel-blocking agents, including nifedipine, nisoldipine, diltiazem, verapamil, CoCl(2) and MnCl(2) . RESULTS: Five micromolar Nifedipine, 1 µM nisoldipine, 10 µM diltiazem, 5 µM verapamil, 3 mM CoCl(2) and 5 mM MnCl(2) evoked uniformly a 90-95% blockade of Ca(2+) current in the absence of isoproterenol. Isoproterenol (100 nM) alone increased the amplitude of Ca(2+) current from 6.8 ± 1.3 to 23.7 ± 2.2 pA/pF in a reversible manner. Isoproterenol caused a marked enhancement of Ca(2+) current even in the presence of nifedipine, nisoldipine, diltiazem and verapamil, but not in the presence of CoCl(2) or MnCl(2) . CONCLUSION: The results indicate that the action of isoproterenol is different in the presence of organic and inorganic Ca(2+) channel blockers. CoCl(2) and MnCl(2) were able to fully prevent the effect of isoproterenol on Ca(2+) current, while the organic Ca(2+) channel blockers failed to do so. This has to be born in mind when the effects of organic Ca(2+) channel blockers are evaluated either experimentally or clinically under conditions of increased sympathetic tone.

Structure-activity relationships of vanilloid receptor agonists for arteriolar TRPV1.

BACKGROUND AND PURPOSE: The transient receptor potential vanilloid 1 (TRPV1) plays a role in the activation of sensory neurons by various painful stimuli and is a therapeutic target. However, functional TRPV1 that affect microvascular diameter are also expressed in peripheral arteries and we attempted to characterize this receptor. EXPERIMENTAL APPROACH: Sensory TRPV1 activation was measured in rats by use of an eye wiping assay. Arteriolar TRPV1-mediated smooth muscle specific responses (arteriolar diameter, changes in intracellular Ca(2+)) were determined in isolated, pressurized skeletal muscle arterioles obtained from the rat and wild-type or TRPV1(-/-) mice and in canine isolated smooth muscle cells. The vascular pharmacology of the TRPV1 agonists (potency, efficacy, kinetics of action and receptor desensitization) was determined in rat isolated skeletal muscle arteries. KEY RESULTS: Capsaicin evoked a constrictor response in isolated arteries similar to that mediated by noradrenaline, this was absent in arteries from TRPV1 knockout mice and competitively inhibited by TRPV1 antagonist AMG9810. Capsaicin increased intracellular Ca(2+) in the arteriolar wall and in isolated smooth muscle cells. The TRPV1 agonists evoked similar vascular constrictions (MSK-195 and JYL-79) or were without effect (resiniferatoxin and JYL-273), although all increased the number of responses (sensory activation) in the eye wiping assay. Maximal doses of all agonists induced complete desensitization (tachyphylaxis) of arteriolar TRPV1 (with the exception of capsaicin). Responses to the partial agonist JYL-1511 suggested 10% TRPV1 activation is sufficient to evoke vascular tachyphylaxis without sensory activation. CONCLUSIONS AND IMPLICATIONS: Arteriolar TRPV1 have different pharmacological properties from those located on sensory neurons in the rat.

Powerful technique to test selectivity of agents acting on cardiac ion channels: the action potential voltage-clamp.

Action potential voltage-clamp (APVC) is a technique to visualize the profile of various currents during the cardiac action potential. This review summarizes potential applications and limitations of APVC, the properties of the most important ion currents in nodal, atrial, and ventricular cardiomyocytes. Accordingly, the profiles ("fingerprints") of the major ion currents in canine ventricular myocytes, i.e. in cells of a species having action potential morphology and set of underlying ion currents very similar to those found in the human heart, are discussed in details. The degree of selectivity of various compounds, which is known to be a critical property of drugs used in APVC experiments, is overviewed. Thus the specificity of agents known to block sodium (tetrodotoxin, saxitoxin), potassium (chromanol 293B, HMR 1556, E-4031, dofetilide, sotalol, 4-aminopyridine, BaCl(2)), calcium (nifedipine, nisolpidine, nicardipine, diltiazem, verapamil, gallopamil), and chloride (anthracene-9-carboxylic acid, DIDS) channels, the inhibitor of the sodium-calcium exchanger (SEA0400), and the activator of sodium current (veratridine) are accordingly discussed. Based on a theory explaining how calcium current inhibitors block calcium channels, the structural comparison of the studied substances usually confirmed the results of the literature. Using these predictions, a hypothetical super-selective calcium channel inhibitor structure was designed. APVC is a valuable tool not only for studying the selectivity of the known ion channel blockers, but is also suitable for safety studies to exclude cardiac ion channel actions of any agent under development.

Long term regulation of cardiac L-type calcium channel by small G proteins.

Calcium ions are crucial elements of excitation-contraction coupling in cardiac myocytes. The intracellular Ca(2+ ) concentration changes continously during the cardiac cycle, but the Ca(2+ ) entering to the cell serves as an intracellular second messenger, as well. The Ca(2+ ) as a second messenger influences the activity of many intracellular signalling pathways and regulates gene expression. In cardiac myocytes the major pathway for Ca(2+ ) entry into cells is L-type calcium channel (LTCC). The precise control of LTCC function is essential for maintaining the calcium homeostasis of cardiac myocytes. Dysregulation of LTCC may result in different diseases like cardiac hypertrophy, arrhytmias, heart failure. The physiological and pathological structural changes in the heart are induced in part by small G proteins. These proteins are involved in wide spectrum of cell biological functions including protein transport, regulation of cell proliferation, migration, apoptosis, and cytoskeletal rearrangement. Understanding the crosstalk between small G proteins and LTCC may help to understand the pathomechanism of different cardiac diseases and to develop a new generation of genetically-encoded Ca(2+ ) channel inhibitors.