Nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated calcium signaling and arrhythmias in the heart evoked by ß-adrenergic stimulation.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca(2+) release in cardiac myocytes evoked by ß-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca(2+) signals sensitive to inhibitors of both acidic Ca(2+) stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca(2+) transients caused by high concentrations of the ß-adrenergic agonist isoproterenol. Ca(2+) transients were recorded both as increases of the free cytosolic Ca(2+) concentration and as decreases of the sarcoplasmic luminal Ca(2+) concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca(2+) transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy.

Novel nucleobase-simplified cyclic ADP-ribose analogue: A concise synthesis and Ca(2+)-mobilizing activity in T-lymphocytes.

A purine nucleobase-simplified cyclic ADP ribose (cADPR) analogue was synthesized, in which a 1,2,3-triazole-4-amide was constructed, instead of a purine moiety, and the northern ribose was replaced by an ether strand. Compound exhibits calcium release activity in intact T-lymphocytes and indicates that it is a membrane-permeable cADPR mimic. Thus, the cADPR analogue containing 1,2,3-triazole-4-amide provides a novel template for further designing cADPR analogues and elucidating their structure-activity relationships.

8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist.

cADPR (cyclic ADP-ribose) is a universal Ca(2+) mobilizing second messenger. In T-cells cADPR is involved in sustained Ca(2+) release and also in Ca(2+) entry. Potential mechanisms for the latter include either capacitative Ca(2+) entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N(1)-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N(1)-cIDPR evoked Ca(2+) signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca(2+) signalling induced by 8-Br-N(1)-cIDPR consisted of Ca(2+) release and Ca(2+) entry. Whereas Ca(2+) release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca(2+) entry was inhibited by the Ca(2+) entry blockers Gd(3+) (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca(2+) entry evoked by 8-Br-N(1)-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H(2)O(2) was enhanced dramatically in those cells, Ca(2+) signalling induced by 8-Br-N(1)-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N(1)-cIDPR. In summary, the sensitivity to the Ca(2+) entry blockers Gd(3+) and SKF-96365 is in favour of the concept of capacitative Ca(2+) entry, secondary to store depletion by 8-Br-N(1)-cIDPR. Taken together, 8-Br-N(1)-cIDPR appears to be the first cADPR agonist affecting Ca(2+) release and secondary Ca(2+) entry, but without effect on TRPM2.

Structural requirements of human ether-a-go-go-related gene channels for block by bupivacaine.

BACKGROUND: Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine. METHODS: Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels. RESULTS: Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 microm (n = 33). Bupivacaine (300 microm) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 microm) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001). CONCLUSIONS: The authors' results indicate that not only the aromatic residues Y652 and F656 but also residues residing deeper within the pore and close to the selectivity filter of HERG channels are involved in inhibition of HERG channels by the low-affinity blocker bupivacaine.

Molecular interaction of droperidol with human ether-a-go-go-related gene channels: prolongation of action potential duration without inducing early afterdepolarization.

BACKGROUND: The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. METHODS: Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. RESULTS: Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 microM. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nM prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. CONCLUSIONS: Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.

Chemotaxis of mouse bone marrow neutrophils and dendritic cells is controlled by adp-ribose, the major product generated by the CD38 enzyme reaction.

The ectoenzyme CD38 catalyzes the production of cyclic ADP-ribose (cADPR) and ADP-ribose (ADPR) from its substrate, NAD(+). Both products of the CD38 enzyme reaction play important roles in signal transduction, as cADPR regulates calcium release from intracellular stores and ADPR controls cation entry through the plasma membrane channel TRPM2. We previously demonstrated that CD38 and the cADPR generated by CD38 regulate calcium signaling in leukocytes stimulated with some, but not all, chemokines and controls leukocyte migration to inflammatory sites. However, it is not known whether the other CD38 product, ADPR, also regulates leukocyte trafficking In this study we characterize 8-bromo (8Br)-ADPR, a novel compound that specifically inhibits ADPR-activated cation influx without affecting other key calcium release and entry pathways. Using 8Br-ADPR, we demonstrate that ADPR controls calcium influx and chemotaxis in mouse neutrophils and dendritic cells activated through chemokine receptors that rely on CD38 and cADPR for activity, including mouse FPR1, CXCR4, and CCR7. Furthermore, we show that the calcium and chemotactic responses of leukocytes are not dependent on poly-ADP-ribose polymerase 1 (PARP-1), another potential source of ADPR in some leukocytes. Finally, we demonstrate that NAD(+) analogues specifically block calcium influx and migration of chemokine-stimulated neutrophils without affecting PARP-1-dependent calcium responses. Collectively, these data identify ADPR as a new and important second messenger of mouse neutrophil and dendritic cell migration, suggest that CD38, rather than PARP-1, may be an important source of ADPR in these cells, and indicate that inhibitors of ADPR-gated calcium entry, such as 8Br-ADPR, have the potential to be used as anti-inflammatory agents.

Long QT 1 mutation KCNQ1A344V increases local anesthetic sensitivity of the slowly activating delayed rectifier potassium current.

BACKGROUND: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only a nonspecific but also a specific pharmacogenetic risk factor for anesthesia. METHODS: The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp and whole cell patch clamp recordings. RESULTS: The mutation A344V induced voltage-dependent inactivation in homomeric KCNQ1 channels and shifted the voltage dependence of KCNQ1/KCNE1 channel activation by +30 mV. The mutation furthermore increased the sensitivity of KCNQ1/KCNE1 channels for bupivacaine 22-fold (KCNQ1wt/KCNE1: IC50 = 2,431 +/- 582 microM, n = 20; KCNQ1A344V/KCNE1: IC50 = 110 +/- 9 microM, n = 24). Pharmacologic effects of the mutant channels were dominant when mutant and wild-type channels were coexpressed. Simulation of cardiac action potentials with the Luo-Rudy model yielded a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication. CONCLUSIONS: The results indicate that certain forms of the LQTS may constitute a specific pharmacogenetic risk factor for regional anesthesia.

Local anesthetic interaction with human ether-a-go-go-related gene (HERG) channels: role of aromatic amino acids Y652 and F656.

BACKGROUND: Human ether-a-go-go-related gene (HERG) potassium channels constitute a potential target involved in cardiotoxic side effects of amino-amide local anesthetics. The molecular interaction site of these low-affinity blockers with HERG channels is currently unknown. The aim of this study was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine. METHODS: The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature. RESULTS: Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4-30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration-response data were described by Hill functions (bupivacaine: wild-type IC50 = 22 +/- 2 microm, n = 38; Y652A IC50 = 95 +/- 5 microm, n = 31). The mutations resulted in a change of the stereoselectivity of HERG channel block by ropivacaine. The potency of the local anesthetics to inhibit wild-type and mutant channels correlated with the lipophilicity of the drug (r > 0.9). CONCLUSIONS: These results indicate that local anesthetics specifically but not exclusively interact with the aromatic residues Y652 and F656 in S6 of HERG channels.

Inhibition of Kv4.3/KChIP2.2 channels by bupivacaine and its modulation by the pore mutation Kv4.3V401I.

BACKGROUND: The transient outward current Ito is an important repolarizing K current in human ventricular myocardium mediated by Kv4.3 and KChIP2.2 subunits. Inhibition of Ito by amino-amide local anesthetics may be involved in severe cardiotoxic side effects. This study elucidates the molecular mechanisms of bupivacaine interaction with complexes formed by Kv4.3 and KChIP2.2 as well as the modulatory effect of KChIP2.2. For this purpose, the pharmacologic effects of bupivacaine on Kv4.3wt/KChIP2.2 channels and on the pore mutant Kv4.3V401I were investigated. METHODS: Kv4.3/KChIP2.2 cDNA was transiently expressed in Chinese hamster ovary cells. Site-directed mutagenesis and patch clamp experiments were performed to analyze the effects of bupivacaine on wild-type and mutant channels. RESULTS: Inhibition of Kv4.3wt/KChIP2.2 channels by bupivacaine was concentration-dependent and reversible. The IC50s for inhibition of the charge conducted by Kv4.3wt/KChIP2.2 channels by bupivacaine and levobupivacaine were 55 +/- 8 and 50 +/- 5 microm, respectively. The local anesthetic accelerated macroscopic current decline of Kv4.3wt/KChIP2.2 and slowed recovery from inactivation without altering steady state inactivation. KChIP2.2 altered the response of Kv4.3wt channels to bupivacaine and bupivacaine modulated KChIP2.2 effects on Kv4.3wt channels. The pore mutation V401I slowed macroscopic current decline of Kv4.3 channels and recovery from inactivation, and it diminished modulation of gating by KChIP2.2. Bupivacaine inhibition of Kv4.3V401I resembled Kv4.3wt and was not changed by coexpression of KChIP2.2. CONCLUSIONS: These results indicate that bupivacaine blocks Kv4.3/KChIP2.2 channels from the open state. They furthermore give structural evidence that amino-amide local anesthetics interfere with the effects of KChIP2.2 on Kv4.3 by an indirect mechanism.

How to build a myofibril.

Building a myofibril from its component proteins requires the interactions of many different proteins in a process whose details are not understood. Several models have been proposed to provide a framework for understanding the increasing data on new myofibrillar proteins and their localizations during muscle development. In this article we discuss four current models that seek to explain how the assembly occurs in vertebrate cross-striated muscles. The models hypothesize: (a) stress fiber-like structures as templates for the assembly of myofibrils, (b) assembly in which the actin filaments and Z-bands form subunits independently from A-band subunits, with the two subsequently joined together to form a myofibril, (c) premyofibrils as precursors of myofibrils, or (d) assembly occurring without any intermediary structures. The premyofibril model, proposed by the authors, is discussed in more detail as it could explain myofibrillogenesis under a variety of different conditions: in ovo, in explants, and in tissue culture studies on cardiac and skeletal muscles.

Myofibrillogenesis in skeletal muscle cells in the presence of taxol.

We address the controversy of whether mature myofibrils can form in the presence of taxol, a microtubule-stabilizing compound. Previous electron microscopic studies reported the absence of actin filaments and Z-bands in taxol-treated myocytes [Antin et al., 1981: J Cell Biol 90:300-308; Toyoma et al., 1982: Proc Natl Acad Sci USA 79:6556-6560]. Quail skeletal myoblasts were isolated from 10-day-old embryos and grown in the presence or absence of taxol. Taxol inhibited the formation of multinucleated elongated myotubes. Myocytes cultured in the continual presence of taxol progressed from rounded to stellate shapes. Groups of myocytes that were clustered together after the isolation procedure fused in the presence of taxol but did not form elongated myotubes. Actin filaments and actin-binding proteins were detected with several different fluorescent probes in all myofibrils that formed in the presence of taxol. The Z-bands contained both alpha-actinin and titin, and the typical arrays of A-Bands were always associated with actin filaments in the myofibrils. Myofibril formation was followed by fixing cells each day in culture and staining with probes for actin, muscle-specific alpha-actinin, myosin II, nebulin, troponin, tropomyosin, and non-muscle myosin II. Small linear aggregates of alpha-actinin or Z-bodies, premyofibrils, were detected at the edges of the myocytes and in the arms of the taxol-treated cells and were always associated with actin filaments. Non-muscle myosin II was detected at the edges of the taxol-treated cells. Removal of the taxol drug led to the cells assuming a normal compact elongated shape. During the recovery process, additional myofibrils formed at the spreading edges of these elongated and thicker myotubes. Staining of these taxol-recovering cells with specific fluorescent reagents reveals three different classes of actin fibers. These results are consistent with a model of myofibrillogenesis that involves the transition of premyofibrils to mature myofibrils.