Marcella Rocchetti

CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation.

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors. Individual TLRs recruit diverse combinations of adaptor proteins, triggering signal transduction pathways and leading to the activation of various transcription factors, including nuclear factor κB, activation protein 1 and interferon regulatory factors. Interleukin-2 is one of the molecules produced by mouse dendritic cells after stimulation by different pattern recognition receptor agonists. By analogy with the events after T-cell receptor engagement leading to interleukin-2 production, it is therefore plausible that the stimulation of TLRs on dendritic cells may lead to activation of the Ca2+/calcineurin and NFAT (nuclear factor of activated T cells) pathway. Here we show that mouse dendritic cell stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase Cγ2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We also show that LPS-induced NFAT activation via CD14 is necessary to cause the apoptotic death of terminally differentiated dendritic cells, an event that is essential for maintaining self-tolerance and preventing autoimmunity. Consequently, blocking this pathway in vivo causes prolonged dendritic cell survival and an increase in T-cell priming capability. Our findings reveal novel aspects of molecular signalling triggered by LPS in dendritic cells, and identify a new role for CD14: the regulation of the dendritic cell life cycle through NFAT activation. Given the involvement of CD14 in disease, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via CD14 is an important step towards the development of potential treatments involving interference with CD14 functions.

A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether‐a‐go‐go‐related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long‐QT syndrome and Drosophila ‘seizure’ mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50 = 16±1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure‐function relationships and novel pharmacological tools with potential therapeutic efficacy.—Gurrola, G. B., Rosati, B., Rocchetti, M., Pimienta, G., Zaza, A., Arcangeli, A., Olivotto, M., Possani, L. D., Wanke, E. A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom. FASEB J. 13, 953–962 (1999)

Rate dependency of delayed rectifier currents during the guinea‐pig ventricular action potential

1 The action potential clamp technique was exploited to evaluate the rate dependency of delayed rectifier currents (IKr and IKs) during physiological electrical activity. IKr and IKs were measured in guinea‐pig ventricular myocytes at pacing cycle lengths (CL) of 1000 and 250 ms. 2 A shorter CL, with the attendant changes in action potential shape, was associated with earlier activation and increased magnitude of both IKr and IKs. Nonetheless, the relative contributions of IKr and IKs to total transmembrane current were independent of CL. 3 Shortening of diastolic interval only (constant action potential shape) enhanced IKs, but not IKr. 4 I Kr was increased by a change in the action potential shape only (constant diastolic interval). 5 In ramp clamp experiments, IKr amplitude was directly proportional to repolarization rate at values within the low physiological range (< 1.0 V s−1); at higher repolarization rates proportionality became shallower and finally reversed. 6 When action potential duration (APD) was modulated by constant current injection (I‐clamp), repolarization rates > 1.0 V s−1 were associated with a reduced effect of IKr block on APD. The effect of changes in repolarization rate was independent of CL and occurred in the presence of IKs blockade. 7 In spite of its complexity, the behaviour of IKr was accurately predicted by a numerical model based entirely on known kinetic properties of the current. 8 Both IKr and IKs may be increased at fast heart rates, but this may occur through completely different mechanisms. The mechanisms identified are such as to contribute to abnormal rate dependency of repolarization in prolonged repolarization syndromes.

Ionic currents during sustained pacemaker activity in rabbit sino-atrial myocytes

1 The contribution of various ionic currents to diastolic depolarization (DD) in rabbit sino‐atrial myocytes was evaluated by the action potential clamp technique. Individual currents were identified, during sustained pacemaking activity reproduced under voltage clamp conditions, according to their sensitivity to supecific channel blockers. 2 The current sensitive to dihydropyridines (DHPs), blockers of L‐type Ca2+ current (ICa,L), was small and outward during most of DD. Diastolic DHP‐sensitive current was affected by changes in the driving force for K+, but it was insensitive to E‐4031, which blocks the current termed IK,r; it was abolished by cell dialysis with a Ca2+ chelator. 3 The current sensitive to 2 mm Cs+ (ICs), a blocker of hyperpolarization‐activated current (If), was inward during the whole DD and it was substantially larger than the net inward current flowing during this phase. However, diastolic IK,r, identified in the same cells as the current sensitive to the blocker E‐4031, exceeded ICs 2‐fold. 4 These findings suggest that: (a) Ca2+ influx during the pacemaker cycle increases a K+ conductance, thus inverting the direction of the net current generated by L‐type Ca2+ channel activity during DD; (b) the magnitude of If would be adequate to account fully for DD; however, the coexistence of a larger IK,r suggests that other channels besides If contribute inward current during this phase.

Dynamic Ca2+-Induced Inward Rectification of K+ Current During the Ventricular Action Potential

Inward rectification, an important determinant of cell excitability, can result from channel blockade by intracellular cations, including Ca2+. However, mostly on the basis of indirect arguments, Ca2+-mediated rectification of inward rectifier K+ current (IK1) is claimed to play no role in the mammalian heart. The present study investigates Ca2+-mediated IK1 rectification during the mammalian ventricular action potential. Guinea pig ventricular myocytes were patch-clamped in the whole-cell configuration. The action potential waveform was recorded and then applied to reproduce normal excitation under voltage-clamp conditions. Subtraction currents obtained during blockade of K+ currents by either 1 mmol/L Ba2+ (IBa) or K+-free solution (I0K) were used to estimate IK1. Similar time courses were observed for IBa and I0K; both currents were strongly reduced during depolarization (inward rectification). Blockade of L-type Ca2+ current by dihydropyridines (DHPs) increased systolic IBa and I0K by 50.7% and 254.5%, respectively. beta-Adrenergic stimulation, when tested on I0K, had an opposite effect; ie, it reduced this current by 66.5%. Ryanodine, an inhibitor of sarcoplasmic Ca2+ release, increased systolic IBa by 47.7%, with effects similar to those of DHPs. Intracellular Ca2+ buffering (BAPTA-AM) increased systolic IBa by 87.7% and blunted the effect of DHPs. Thus, IK1 may be significantly reduced by physiological Ca2+ transients determined by both Ca2+ influx and release. Although Ca2+-induced effects may represent only a small fraction of total IK1 rectification, they are large enough to affect excitability and repolarization. They may also contribute to facilitation of early afterdepolarizations by conditions increasing Ca2+ influx.

Sulfonylureas blockade of neural and cardiac HERG channels

The human ether‐a‐go‐go‐related gene (herg) encodes a K+ current (I HERG) which plays a fundamental role in heart excitability and in neurons by contributing to action potential repolarization and to spike‐frequency adaptation, respectively. In this paper we show that I HERG, recorded in neuroblastoma cells and guinea‐pig ventricular myocytes, was reversibly inhibited by the KATP channel blocker glibenclamide (IC50=74 μM). The voltage and use dependence of glibenclamide blockade were also evaluated. Another sulfonylurea, glimepiride, had less effective results in blocking I HERG. The findings of this study are relevant to the interpretation of glibenclamide effects on cellular electrophysiology and suggest that oral antidiabetic therapy with sulfonylureas may contribute to iatrogenic QT prolongation and related arrhythmias.

Modulation of the Hyperpolarization-Activated Current (If) by Adenosine in Rabbit Sinoatrial Myocytes

BACKGROUND Modulation of sinoatrial pacemaking by adenosine (Ado) in the absence of concomitant adrenergic stimulation (direct modulation) has been attributed to activation of a K+ conductance. In the present study, we evaluated the direct effects of Ado on the pacemaking current I(f) and tested their interaction with those of acetylcholine (ACh). METHODS AND RESULTS Rabbit sinoatrial myocytes were patch-clamped at 35 degrees C in the presence of 1 mmol/L BaCl2 and 2 mmol/ L MnCl2, Ado (1 mumol/L) reversibly reduced I(f) by 33.1 +/- 5.7% of control (n = 5; P < .05). Ado (1 mumol/L) reversibly shifted I(f) midactivation potential by -6.63 +/- 1.18 mV (n = 4; P < .05). Fully activated I(f) conductance (0.262 +/- 0.037 versus 0.254 +/- 0.036 nS/ pF; n = 6, NS) and reversal potential (-17.35 +/- 0.99 versus -18.01 +/- 1.42 mV; n = 6, NS) were not changed by 10 mumol/L Ado. The Ado receptor antagonist 8-PST (10 mumol/L) reversed the effect of 0.3 mumol/L Ado by 64.9 +/- 4.2% (n = 6; P < .05). Ado maximally shifted the I(f) activation curve by -5.85 mV, with a half-maximal concentration of 0.0796 mumol/L (n = 93). The shifts in I(f) activation induced by Ado (0.3 mumol/L) and ACh (1 mumol/ L) separately were -4.89 +/- 0.05 and -8.84 +/- 0.51 mV, respectively; concomitant Ado and ACh superfusion shifted activation by -9.7 +/- 0.45 mV (NS versus ACh alone; n = 9). Threshold Ado concentrations dose-dependently reduced the rate of spontaneous pacemaker activity (eg, -18.8 +/- 3.4% at Ado 0.03 mumol/L). CONCLUSIONS Submicromolar Ado directly inhibits I(f) and slows pacemaking in sinoatrial myocytes; the mode of I(f) inhibition is similar to that previously described for ACh. Thus, Ado may exert local modulation of sinus rate through signaling pathways similar to those used by ACh.

Role of the input/output relation of sinoatrial myocytes in cholinergic modulation of heart rate variability.

Input/Output Relation of the Sinoatrial Node. Introduction: Modulation of sinus rate may be viewed as the transduction of an input signal (receptor stimulation) into an output signal (cycle length [CL]) by the sinus node. This study analyzes the input/output (I/O) relation of sinoatrial pacemaking elements and tests its impact on cholinergic modulation of heart rate variability.

Rate dependency of β‐adrenergic modulation of repolarizing currents in the guinea‐pig ventricle

β‐Adrenergic stimulation modulates ventricular currents and sinus cycle length (CL). We investigated how changes in CL affect the current induced by isoprenaline (Iso) during the action potential (AP) of guinea‐pig ventricular myocytes. Action‐potential clamp was applied at CLs of 250 and 1000 ms to measure: (1) the net current induced by 0.1 μm Iso (IIso); (2) the L‐type Ca2+ current ICaL and slow delayed rectifier current IKs components of IIso (IIsoCa and IIsoK), identified as the Iso‐induced current sensitive to nifedipine and HMR1556, respectively; and (3) IIso persisting after inhibition of both ICa and IKs (IisoR). The pause dependency of IKs and its modulation were evaluated in voltage‐clamp experiments. The rate dependency of the duration of the action potential at 90% repolarization (APD90) and its modulation by isoprenaline were tested in current‐clamp experiments. At a CL of 250 ms IIso was inward during initial repolarization and reversed at 59% of APD90. At a CL of 1000 ms IIso became mostly inward in all cells. Switching to shorter CL did not change IIsoCa and IIsoK amplitudes, but moved their peak amplitudes to earlier repolarization; IIsoR was independent of CL. Acceleration of IIsoK at shorter CL was based on faster pause dependency of IKs activation rate. The ‘restitution’ of activation rates was modulated by isoprenaline. The APD90–CL relation was rotated anticlockwise by isoprenaline and crossed the control curve at a CL of 150 ms (400 beats min−1). We conclude that: (1) isoprenaline induced markedly different current profiles according to pacing rate, involving CL‐dependent ICa and IKs modulation; (2) the effect of isoprenaline on APD90 was CL dependent, and negligible during tachycardia; and (3) during sympathetic activation, repolarization stability may involve matched modulation of sinus rate and repolarizing currents.

The late Na+ current--origin and pathophysiological relevance

In excitable tissues, voltage-dependent Na+ current (INa) is best known for supporting autoregenerative depolarization and impulse propagation. Its transient component (INaT), which is large and terminated within several milliseconds by channel inactivation, fulfils this role. Nevertheless, INa also includes a smaller sustained component, i.e. one persisting during prolonged membrane depolarization, which contributes to repolarization course. Sustained INa implies slow or incomplete inactivation of a proportion of the Na+ channels activated during the action potential upstroke. Several mechanisms may underlie this phenomenon and contribute to arrhythmogenesis in different conditions.