Julio L. Alvarez

Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channels

Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins. We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660u2003nM and modify the resting potentials. The effect on Na+ currents in rat cardiomyocytes was studied using the patch‐clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78u2003nM, respectively. On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8u2003μM), shift the steady‐state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials. The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins. We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady‐state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels.

Zinc modulation of basal and β-adrenergically stimulated L-type Ca2+ current in rat ventricular cardiomyocytes: consequences in cardiac diseases.

Zinc exists in biological systems as bound and histochemically reactive free Zn2+ in the nanomolar range. Zinc is required as either structural or catalytic component for a large number of enzymes. It also modulates current passage through many ion channels. Here, we reinvestigated the effects of extracellular and intracellular Zn2+ on the L-type Ca2+ current (ICaL) and its modulation by β-adrenergic stimulation in rat ventricular cardiomyocytes. In the absence of Ca2+ ions, Zn2+ could permeate through the L-type channel at much lower concentrations and at a more positive voltage range, but with a lower permeability than Ca2+. In the presence of Ca2+, extracellular Zn2+ demonstrated strong bimodal inhibitory effects on the ICaL, with half-inhibition occurring around 30xa0nM, i.e., in the range of concentrations found in the plasma. Intracellular Zn2+ also significantly inhibited the ICaL with a half-inhibitory effect at 12.7xa0nM. Moreover, β-adrenergic stimulation was markedly reduced by intracellular Zn2+ at even lower concentrations (<1xa0nM) as a consequence of Zn2+-induced inhibition of the adenylyl cyclase. All these effects appeared independent of redox variations and were not affected by dithiothreitol. Thus, both basal intracellular and extracellular Zn2+ modulate transmembrane Ca2+ movements and their regulation by β-adrenergic stimulation. Considering that, in many pathological situations, including diabetes, the extracellular Zn2+ concentration is reduced and the intracellular one is increased, our results help to explain both Ca2+ overload and marked reduction in the β-adrenergic stimulation in these diseases.

Proarrhythmic effect of sustained EPAC activation on TRPC3/4 in rat ventricular cardiomyocytes

The Exchange Protein directly Activated by cAMP (EPAC) participates to the pathological signaling of cardiac hypertrophy and heart failure, in which the role of Ca(2+) entry through the Transient Receptor Potential Canonical (TRPC) channels begin to be appreciated. Here we studied whether EPAC activation could influence the activity and/or expression of TRPC channels in cardiac myocytes. In adult rat ventricular myocytes treated for 4 to 6h with the selective EPAC activator, 8-pCPT (10μM), we observed by Fluo-3 confocal fluorescence a Store-Operated Ca(2+) Entry (SOCE) like-activity, which was blunted by co-incubation with EPAC inhibitors (ESI-05 and CE3F4 at 10 μM). This SOCE-like activity, which was very small in control incubated cells, was sensitive to 30-μM SKF-96365. Molecular screening showed a specific upregulation of TRPC3 and C4 protein isoforms after 8-pCPT treatment. Moreover, sustained EPAC activation favored proarrhythmic Ca(2+) waves, which were reduced either by co-incubation with EPAC inhibitors or bath perfusion with TRPC inhibitors. Our study provides the first evidence that sustained selective EPAC activation leads to an increase in TRPC3 and C4 protein expression and induces a proarrhythmic SOCE-like activity in adult rat ventricular cardiomyocytes, which might be of importance during the development of cardiac diseases.

Effects of a toxin from the mucus of the caribbean sea anemone (Bunodosoma granulifera) on the ionic currents of single ventricular mammalian cardiomyocytes

The effects were studied of a toxin (Bainh) isolated from the secretion of the Caribbean sea anemone Bunodosoma granulifera on electrical and mechanical activities of rat ventricular muscle. The effects on the ionic currents of single rat and dog ventricular cardiomyocytes were studied using the whole-cell recording patch-clamp technique. In the concentration range from 1 to 10 mg/ml, Bainh increased the force of contraction and induced an increase in action potential duration of ventricular multicellular preparations. In single cardiomyocytes, at concentrations up to 10 mg/ml Bainh showed no significant effects on the sodium current. However, at 0.5-1 mg/ml it increased the L-type Ca current (ICaL) by 25-50%. This increase in ICaL was not voltage dependent and was reversible after washout. The transient outward current was not significantly affected by Bainh (1-10 mg/ml). In this concentration range, Bainh markedly (approximately 75%) increased the inward-going rectifier current, IKI. This effect that was not voltage dependent and was fully reversible upon returning to control solution. It is suggested that these effects on ionic currents could explain the positive inotropic action of Bainh on cardiac multicellular preparations.

TRPV4 activation triggers protective responses to bacterial lipopolysaccharides in airway epithelial cells

Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca2+ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction.LPS is a major component of gram-negative bacterial cell walls, and triggers immune responses in airway epithelium by activating TLR4. Here the authors show that LPS also activates TRPV4, thereby inducing fast defense responses such as nitric oxide production and increased ciliary beating in mice.

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca2+ channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca2+ currents in mouse ventricular cardiomyocytes (VCM, with Ca2+ as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba2+ as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca2+ channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca2+ channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

Cardiac cellular actions of hydrochlorothiazide

In long term treatment, thiazide diuretics such as hydrochlorothiazide (HCTZ) lower blood pressure by decreasing peripheral resistance rather than by their diuretic effect. This action has been attributed to the opening of Ca2+‐activated K+ channels in vascular smooth muscle cells. However, little is known about their cardiac cellular actions. Here we investigated the possible actions of HCTZ on action potential and contraction of rat ventricular muscle strips and on the ionic currents of isolated rat ventricular cardiomyocytes. HCTZ depressed ventricular contraction with an IC30 of 1.85u2003μM (60% decrease at 100u2003μM). Action potential duration at −60u2003mV and maximal rate of depolarization were, however, only slightly decreased by 12% and 22%, respectively, at 100u2003μM. At the single cell level, HCTZ (100u2003μM) depressed the fast Na+ current (INa) and the L‐type Ca2+ current (ICaL) by 30% and 20%, respectively. The effects on ICaL were not voltage‐or frequency‐dependent. In cells intracellularly perfused with 50u2003μM cyclic adenosine, monophosphate HCTZ reduced ICaL by 33%. The transient (Ito), the delayed rectifier and the inward rectifier potassium currents were decreased by 20% at 100u2003μM HCTZ. The effects on Ito were voltage‐dependent. In conclusion, HCTZ at high concentrations possesses a negative inotropic action that could be in part due to its blocking action on INa and ICaL. The actions of HCTZ on multiple cardiac ionic currents could explain its weak effect on action potential duration.

Silica nanoparticles inhibit the cation channel TRPV4 in airway epithelial cells

BackgroundSilica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells.ResultsUsing fluorometric measurements of intracellular Ca2+ concentration ([Ca2+]i) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca2+ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca2+]i, but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells.ConclusionsOur results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.