Leucine-rich repeat containing protein LRRC8A is essential for swelling-activated Cl- currents and embryonic development in zebrafish.

A volume-regulated anion channel (VRAC) has been electrophysiologically characterized in innumerable mammalian cell types. VRAC is activated by cell swelling and mediates the volume regulatory efflux of Cl(-) and small organic solutes from cells. Two groups recently identified the mammalian leucine-rich repeat containing protein LRRC8A as an essential VRAC component. LRRC8A must be coexpressed with at least one of the other four members of this gene family, LRRC8B-E, to reconstitute VRAC activity in LRRC8(-/-) cells. LRRC8 genes likely arose with the origin of chordates. We identified LRRC8A and LRRC8C-E orthologs in the zebrafish genome and demonstrate that zebrafish embryo cells and differentiated adult cell types express a swelling-activated Cl(-) current indistinguishable from mammalian VRAC currents. Embryo cell VRAC currents are virtually eliminated by morpholino knockdown of the zebrafish LRRC8A ortholog lrrc8aa VRAC activity is fully reconstituted in LRRC8(-/-) human cells by coexpression of zebrafish lrrc8aa and human LRRC8C cDNAs. lrrc8aa expression varies during zebrafish embryogenesis and lrrc8aa knockdown causes pericardial edema and defects in trunk elongation and somatogenesis. Our studies provide confirmation of the importance of LRRC8A in VRAC activity and establish the zebrafish as a model system for characterizing the molecular regulation and physiological roles of VRAC and LRRC8 proteins.

Impaired heart rate regulation and depression of cardiac chronotropic and dromotropic function in polymicrobial sepsis.

The scope of cardiac pathophysiology in sepsis has not been fully defined. Accordingly, we evaluated the effects of sepsis on heart rate (HR), HR variability, and conduction parameters in a murine model of sepsis. Electrocardiograms were recorded noninvasively from conscious mice before and after cecal ligation and puncture (CLP) or sham surgery. Responses of isolated atria to tyramine and isoproterenol were quantified to assess the functional state of sympathetic nerves and postjunctional sensitivity to adrenergic stimulation. Cecal ligation and puncture mice had lower HR compared with sham at 16 to 18 h postsurgery (sham, 741 ± 7 beats/min; CLP, 557 ± 31 beats/min; n = 6/group; P < 0.001), and there was significant prolongation of the PR, QRS, and QTc intervals. Slowing of HR and conduction developed within 4 to 6 h after CLP and were preceded by a decrease in HR variability. Treatment of CLP mice with isoproterenol (5 mg/kg, intraperitoneally) at 25 h after surgery failed to increase HR or decrease conduction intervals. The lack of in vivo response to isoproterenol cannot be attributed to hypothermia because robust chronotropic and inotropic responses to isoproterenol were evoked from isolated atria at 25 °C and 30 °C. These findings demonstrate that impaired regulation of HR (i.e., reduced HR variability) develops before the onset of overt cardiac rate and conduction changes in septic mice. Subsequent time-dependent decreases in HR and cardiac conduction can be attributed to hypothermia and would contribute to decreased cardiac output and organ perfusion. Because isolated atria from septic mice showed normal responsiveness to adrenergic stimulation, we conclude that impaired effectiveness of isoproterenol in vivo can be attributed to reversible effects of systemic factors on adrenergic receptors and/or postreceptor signaling.

9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes.

It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 µM) and flufenamic acid (10 and 100 µM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca(2+)-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10 µM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

Lipopolysaccharide prolongs action potential duration in HL-1 mouse cardiomyocytes.

Sepsis has deleterious effects on cardiac function including reduced contractility. We have shown previously that lipopolysaccharides (LPS) directly affect HL-1 cardiac myocytes by inhibiting Ca(2+) regulation and by impairing pacemaker "funny" current, I(f). We now explore further cellular mechanisms whereby LPS inhibits excitability in HL-1 cells. LPS (1 µg/ml) derived from Salmonella enteritidis decreased rate of firing of spontaneous action potentials in HL-1 cells, and it increased their pacemaker potential durations and decreased their rates of depolarization, all measured by whole cell current clamp. LPS also increased action potential durations and decreased their amplitude in cells paced at 1 Hz with 0.1 nA, and 20 min were necessary for maximal effect. LPS decreased the amplitude of a rapidly inactivating inward current attributed to Na(+) and of an outward current attributed to K(+); both were measured by whole cell voltage clamp. The K(+) currents displayed a resurgent outward tail current, which is characteristic of the rapid delayed-rectifier K(+) current, I(Kr). LPS accordingly reduced outward currents measured with pipette Cs(+) substituted for K(+) to isolate I(Kr). E-4031 (1 µM) markedly inhibited I(Kr) in HL-1 cells and also increased action potential duration; however, the direct effects of E-4031 occurred minutes faster than the slow effects of LPS. We conclude that LPS increases action potential duration in HL-1 mouse cardiomyocytes by inhibition of I(Kr) and decreases their rate of firing by inhibition of I(Na.) This protracted time course points toward an intermediary metabolic event, which either decreases available mouse ether-a-go-go (mERG) and Na(+) channels or potentiates their inactivation.

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca(2+)](i), I(Ca), and Ca(2+) transients in HL-1 cardiomyocytes.

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca(2+)](i), Ca(2+) transients and membrane Ca(2+) current, I(Ca), in cultured murine HL-1 cardiomyocytes. LY294002 (1-20 µM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca(2+)](i), Ca(2+) transients and I(Ca). We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2-8 nM); ß (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca(2+)](i) regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca(2+)](i), and inhibited Ca(2+) transients. Triciribine (1-20 µM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca(2+)](i), and Ca(2+) transients and I(Ca). We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca(2+)](i) in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca(2+)](i) required for excitation-contraction coupling in cardiomyoctyes.

Benzoquinone ansamycin 17AAG binds to mitochondrial voltage-dependent anion channel and inhibits cell invasion.

Geldanamycin and its derivative 17AAG [17-(Allylamino)-17-demethoxygeldanamycin, telatinib] bind selectively to the Hsp90 chaperone protein and inhibit its function. We discovered that these drugs associate with mitochondria, specifically to the mitochondrial membrane voltage-dependent anion channel (VDAC) via a hydrophobic interaction that is independent of HSP90. In vitro, 17AAG functions as a Ca(2+) mitochondrial regulator similar to benzoquinone-ubiquinones like Ub0. All of these compounds increase intracellular Ca(2+) and diminish the plasma membrane cationic current, inhibiting urokinase activity and cell invasion. In contrast, the HSP90 inhibitor radicicol, lacking a bezoquinone moiety, has no measurable effect on cationic current and is less effective in influencing intercellular Ca(2+) concentration. We conclude that some of the effects of 17-AAG and other ansamycins are due to their effects on VDAC and that this may play a role in their clinical activity.

Lipopolysaccharides directly decrease Ca2+ oscillations and the hyperpolarization-activated nonselective cation current If in immortalized HL-1 cardiomyocytes.

Lipopolysaccharide (LPS) has been implicated in sepsis-mediated heart failure and chronic cardiac myopathies. We determined that LPS directly and reversibly affects cardiac myocyte function by altering regulation of intracellular Ca2+ concentration ([Ca2+]i) in immortalized cardiomyocytes, HL-1 cells. [Ca2+]i oscillated (<0.4 Hz), displaying slow and transient components. LPS (1 microg/ml), derived either from Escherichia coli or from Salmonella enteritidis, reversibly abolished Ca2+ oscillations and decreased basal [Ca2+]i by 30-40 nM. HL-1 cells expressed Toll-like receptors, i.e., TLR-2 and TLR-4. Thus, we differentiated effects of LPS on [Ca2+]i and Ca2+ oscillations by addition of utlrapure LPS, a TLR-4 ligand. Ultrapure LPS had no effect on basal [Ca2+]i, but it reduced the rate of Ca2+ oscillations. Interestingly, Pam3CSK4, a TLR-2 ligand, affected neither Ca2+ parameter, and the effect of ultrapure LPS and Pam3CSK4 combined was similar to that of utlrapure LPS alone. Thus, unpurified LPS directly inhibits HL-1 calcium metabolism via TLR-4 and non-TLR-4-dependent mechanisms. Since others have shown that endotoxin impairs the hyperpolarization-activated, nonselective cationic pacemaker current (I(f)), which is expressed in HL-1 cells, we utilized whole cell voltage-clamp techniques to demonstrate that LPS (1 microg/ml) reduced I(f) in HL-1 cells. This inhibition was marginal at physiologic membrane potentials and significant at very negative potentials (P < 0.05 at -140, -150, and -160 mV). So, we also evaluated effects of LPS on tail currents of fully activated I(f). LPS reduced the slope conductance of the tail currents from 498 +/- 140 pS/pF to 223 +/- 65 pS/pF (P < 0.05) without affecting reversal potential of -11 mV. Ultrapure LPS had similar effect on I(f), whereas Pam3CSK4 had no effect on I(f). We conclude that LPS inhibits activation of I(f), enhances its deactivation, and impairs regulation of [Ca2+]i in HL-1 cardiomyocytes via TLR-4 and other mechanisms.

Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration.

This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 (TRPM8) ion channels, to increase intracellular Ca2+ concentration, [Ca2+]i, in human glioblastoma cells (DBTRG cells), which resulted in activation of the large-conductance Ca2+-activated K+ membrane ion channels (BK channels). Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 microM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 microM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol increased [Ca2+]i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca2+-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell [Ca2+]i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline reverses menthol-stimulated increase of [Ca2+]i and of cell migration. Thus, BK channels function to maintain elevations in [Ca2+]i needed to sustain increases in DBTRG cell migration.

HGF/SF and menthol increase human glioblastoma cell calcium and migration.

This study explored the role of transient receptor potential melastatin 8 ion channels (TRPM8) in mechanisms of human glioblastoma (DBTRG) cell migration. Menthol stimulated influx of Ca(2+), membrane current, and migration of DBTRG cells. Effects on Ca(2+) and migration were enhanced by pre-treatment with hepatocyte growth factor/scatter factor (HGF/SF). Effects on Ca(2+) also were greater in migrating cells compared with non-migrating cells. 2-Aminoethoxydiphenyl borate (2-APB) inhibited all menthol stimulations. RT-PCR and immunoblot analysis showed that DBTRG cells expressed both mRNA and protein for TRPM8 ion channels. Two proteins were evident: one (130-140 kDa) in a plasma membrane-enriched fraction, and a variant (95-100 kDa) in microsome- and plasma membrane-enriched fractions. Thus, TRPM8 plays a role in mechanisms that increase [Ca(2+)](i) needed for DBTRG cell migration.

Phenotypic properties of adult mouse intrinsic cardiac neurons maintained in culture.

Intrinsic cardiac neurons are core elements of a complex neural network that serves as an important integrative center for regulation of cardiac function. Although mouse models are used frequently in cardiovascular research, very little is known about mouse intrinsic cardiac neurons. Accordingly, we have dissociated neurons from adult mouse heart, maintained these cells in culture, and defined their basic phenotypic properties. Neurons in culture were primarily unipolar, and 89% had prominent neurite outgrowth after 3 days (longest neurite length of 258 +/- 20 microm, n = 140). Many neurites formed close appositions with other neurons and nonneuronal cells. Neurite outgrowth was drastically reduced when neurons were kept in culture with a majority of nonneural cells eliminated. This finding suggests that nonneuronal cells release molecules that support neurite outgrowth. All neurons in coculture showed immunoreactivity for a full complement of cholinergic markers, but about 21% also stained for tyrosine hydroxylase, as observed previously in sections of intrinsic cardiac ganglia from mice and humans. Whole cell patch-clamp recordings demonstrated that these neurons have voltage-activated sodium current that is blocked by tetrodotoxin and that neurons exhibit phasic or accommodating patterns of action potential firing during a depolarizing current pulse. Several neurons exhibited a fast inward current mediated by nicotinic ACh receptors. Collectively, this work shows that neurons from adult mouse heart can be maintained in culture and exhibit appropriate phenotypic properties. Accordingly, these cultures provide a viable model for evaluating the physiology, pharmacology, and trophic factor sensitivity of adult mouse cardiac parasympathetic neurons.

TRPV channels and modulation by hepatocyte growth factor/scatter factor in human hepatoblastoma (HepG2) cells.

Using patch clamp and Ca(2+) imaging techniques, we have studied Ca(2+) entry pathways in human hepatoblastoma (HepG2) cells. These cells express the mRNA of TRPV1, TRPV2, TRPV3 and TRPV4 channels, but not those of TRPV5 and TRPV6. Functional assessment showed that capsaicin (10 microM), 4alpha-phorbol-12,13-didecanoate (4alphaPDD, 1 microM), arachidonic acid (10 microM), hypotonic stress, and heat all stimulated increases in [Ca(2+)](i) within minutes. The increase in [Ca(2+)](i) depended on extracellular Ca(2+) and on the transmembrane potential, which indicated that both driving forces affected Ca(2+) entry. Capsaicin also stimulated an increase in [Ca(2+)](i) in nominally Ca(2+)-free solutions, which was compatible with the receptor functioning as a Ca(2+) release channel. Hepatocyte growth factor/scatter factor (HGF/SF) modulated Ca(2+) entry. Ca(2+) influx was greater in HepG2 cells incubated with HGF/SF (20 ng/ml for 20 h) compared with non-stimulated cells, but this occurred only in those cells with a migrating phenotype as determined by presence of a lamellipodium and trailing footplate. The effect of capsaicin on [Ca(2+)](i) was greater in migrating HGF/SF-treated cells, and this was inhibited by capsazepine. The difference between control and HGF/SF-treated cells was not found in Ca(2+)-free solutions. 4alphaPDD also had no greater effect on HGF/SF-treated cells. We conclude that TRPV1 and TRPV4 channels provide Ca(2+) entry pathways in HepG2 cells. HGF/SF increases Ca(2+) entry via TRPV1, but not via TRPV4. This rise in [Ca(2+)](i) may constitute an early response of a signalling cascade that gives rise to cell locomotion and the migratory phenotype.

Modulation of TRPV4 gating by intra- and extracellular Ca2+.

We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4alphaPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+](e)) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca2+](e). It was also much slower in the presence of [Ca2+](e) in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797), were constitutively open. In conclusion, gating of the 4alphaPDD-activated TRPV4 channel depends on both extra- and intracellular Ca2+, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.

Transient receptor potential channels in endothelium: solving the calcium entry puzzle?

Many endothelial cell (EC) functions depend on influx of extracellular Ca2+, which is triggered by a variety of mechanical and chemical signals. Here, we discuss possible pathways for this Ca2+ entry. The superfamily of cation channels derived from the "transient receptor potential" (TRP) channels is introduced. Several members of this family are expressed in ECs, and they provide pathways for Ca2+ entry. All TRP subfamilies may contribute to the Ca2+ entry channels or to the regulation of Ca2+ entry in EC. Members of Ca2+ entry channels in endothelium probably belong to the canonical TRP subfamily, TRPC. All TRPC1-6 have been discussed as Ca2+ entry channels that might be store-operated and/or receptor-operated. More importantly, knockout models of TRPC4 have proven that this channel is functionally involved in the regulation of endothelial-dependent vasorelaxation and in the control of EC barrier function. TRPC1 might be an important candidate for involvement of endothelial growth factors. TRPC3 is unequivocally important for a sustained EC Ca2+ entry. ECs express different patterns of TRPCs, which may increase the variability of TRPC channel function by formation of different multiheteromers. Among the two other TRP subfamilies, TRPMV and TRPM, at least TRPV4 and TRPM4 are EC channels. TRPV4 is a Ca2+ entry channel that is activated by an increase in cell volume, which might be involved in mechano-sensing, by an increase in temperature, and perhaps by ligand-activation. TRPM4 is a nonselective cation channel, which is not Ca2+ permeable. It is probably modulated by NO and might be essential for regulating the inward driving force for Ca2+ entry. Possible modes of TRP channel regulation are described, involving (a) activation via the phospholipase (PL)Cbeta and PLC-gamma pathways; (b) activation by lipids (diacylglycerol [DAG], arachidonic acid); (c) Ca2+ depletion of Ca2+ stores in the endoplasmic reticulum; (d) shear stress; and (e) radicals.