Aldo Giovannelli

CXC chemokines interleukin-8 (IL-8) and growth-related gene product α (GROα) modulate Purkinje neuron activity in mouse cerebellum

Abstract We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROα by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROα may play a neuromodulatory role on mouse cerebellum.

SDF‐1α‐mediated modulation of synaptic transmission in rat cerebellum

The functional expression of the seven‐transmembrane domain G protein‐coupled chemokine receptor CXCR‐4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti‐CXCR‐4 Ab, and by evaluating the calcium responses to the physiological agonist stromal‐derived cell factor‐1α (SDF‐1α) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR‐4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF‐1α, both in cell bodies and in neuronal processes. Whole‐cell patch‐clamped PNs in cerebellar slices responded to SDF‐1α application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF‐1α‐induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR‐4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.

Modulation of the neurotransmitter release in rat cerebellar neurons by GROβ

WE report here that, in cultured cerebellar granule cells, the CXC chemokine GRO β stimulates the signaling pathway of the extracellular signal-regulated kinases, and enhances both evoked and spontaneous post-synaptic currents in patch clamped Purkinje neurons from rat cerebellar slices. The GRO β-induced enhancement of the excitatory post-synaptic currents evoked by stimulating the parallel fibres is blocked by the inhibitor of the extracellular signal-regulated kinases pathway PD98059, which also reduces both basal frequency of spontaneous post-synaptic currents and mean amplitude of evoked excitatory post-synaptic currents. Our results suggest that GRO β modulates neurotransmitter release in the cerebellum through the activation of the extra-cellular signal-regulated kinases pathway.

Stimulation of chemokine CXC receptor 4 induces synaptic depression of evoked parallel fibers inputs onto Purkinje neurons in mouse cerebellum

In the present work, we studied the effects of the stimulation of the chemokine CXC receptor 4 (CXCR4) by the stromal-derived cell growth factor-1alpha (SDF-1alpha) on the evoked excitatory postsynaptic current. This was generated in Purkinje neurons (PN) from mouse cerebellar slices by the stimulation of parallel fibers. It was found that the amplitude of EPSC was reversibly reduced by SDF-1alpha application. This effect was dose-dependent (IC(50)=0.34 nM) and was abolished by the anti-CXCR4 monoclonal antibody (mAb) 12G5. This SDF-1alpha-induced synaptic depression was caused by a decrease of evoked glutamate release, rather than a decrease in the postsynaptic glutamate receptor (GluR) sensitivity, as the mean amplitude of the spontaneous EPSCs was not influenced by chemokine application. Moreover, NMDA receptors (NMDARs) are involved in EPSC depression being inhibited by the NMDAR blocker 2-amino-5-phosphonopentanoic acid (AP-5). The mechanisms by which SDF-1alpha modulates neurotransmission in the cerebellar cortex are discussed.

SPONTANEOUS AND REPETITIVE CALCIUM TRANSIENTS IN C2C12 MOUSE MYOTUBES DURING IN VITRO MYOGENESIS

Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca2+ concentration ([Ca2+]i in the mouse muscle cell line C2C12 during in vitro myogenesis. Three different patterns of changes in [Ca2+]i were observed: (i) [Ca2+]i oscillations; (ii) faster Ca2+ events confined to subcellular regions (localized [Ca2+]i spikes) and (iii) [Ca2+]i spikes detectable in the entire myotube (global [Ca2+]i spikes). [Ca2+]i oscillations and localized [Ca2+]i spikes were detectable following the appearance of caffeine‐sensitivity in differentiating C2C12 cells. Global [Ca2+]i spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage‐operated Ca2+ channels and ryanodine receptors. In contrast to [Ca2+]i oscillations and localized [Ca2+]i spikes, the global events immediately stopped when cells were perfused either with a Ca2+‐free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca2+]i spikes, membrane currents and fluorescence signals were measured simultaneously. These experiments revealed that global [Ca2+]i spikes were correlated with an inward current. Moreover, while the depletion of the Ca2+ stores blocked [Ca2+]i oscillations and localized [Ca2]i spikes, it only reduced the amplitude of global [Ca2+]i spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca2+]i changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage‐operated Ca2+ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca2+]i spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.

Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)‐induced single‐channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α3β4, chick α<3/β2, α4β2, α4β4, α7and α8, expression levels were high enough to allow measurements of acetylcholine‐evoked whole‐cell currents and nicotine‐elicited Ca2+ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine‐evoked events of α8 nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α7 and α8 nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α3α4nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α3α4and α3α2) exhibited single‐channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.

Activation of the nicotinic acetylcholine receptor mobilizes calcium from caffeine-insensitive stores in C2C12 mouse myotubes

In cultured mouse C2C12 myotubes, digital Ca2+ imaging fluorescence microscopy using the acetoxymethyl ester of Fura-2, Fura-2-AM, showed that, in the absence of extracellular Ca2+, acetylcholine (ACh) and nicotine, but not muscarine, raised the intracellular concentration of Ca2+ ([Ca2+]i) by about tenfold. AChinduced Ca2+ mobilization was prevented by thapsigargin, a drug known to deplete inositol 1,4,5-trisphosphate (InsP3)-sensitive stores, and was concomitant with InsP3 accumulation. Caffeine, which releases Ca2+ from the ryanodine-sensitive stores of the sarcoplasmic reticulum, did not interfere with the ACh-induced [Ca2+]i increase. Ca2+ mobilization was also inhibited when myotubes were depolarized by high K+, or when extracellular Na+ was omitted. Nicotinic ACh receptor (nAChR) stimulation lowered intracellular pH with a time course slower than the [Ca2+]i increase. Possible mechanisms linking the current flowing through the nAChR pore to [Ca2+]i increase are discussed.

The growth-related gene product β induces sphingomyelin hydrolysis and activation of c-Jun N-terminal kinase in rat cerebellar granule neurones

The growth-related gene product β (GROβ) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and GROβ receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express the GROβ receptor CXCR2. We also show that, in addition to the known stimulation of a phosphoinositide-specific phospholipase C, GROβ activates both neutral (N-) and acidic (A-) sphingomyelinases (SMase) and the stress-activated c-Jun N-terminal kinase 1 (JNK1). Although both exogenous ceramide and bacterial SMase stimulate JNK1, GROβ-induced JNK1 activation is an event probably independent of ceramide generated by A-SMase, since it is maintained in the presence of compounds that block A-SMase activity. This is the first report on the activation of the SMase pathway by chemokines.

Zinc permeates mouse muscle ACh receptor channels expressed in BOSC 23 cells and affects channel function

1 The influx of Zn2+ through the channels of fetal and adult mouse muscle nicotinic acetylcholine receptors (γ‐ and ε‐AChRs) and its effects on receptor function were studied in transiently transfected human BOSC 23 cells, by combining patch‐clamp recordings with digital fluorescence microscopy. 2 ACh‐induced whole‐cell currents were reversibly reduced by external ZnCl2, with half‐maximal inhibitory concentrations of 3 and 1 mM for γ‐ and ε‐AChRs, respectively. 3 Both γ‐ and ε‐AChR channels were permeable to Zn2+, as shown by fluorescence measurements using Zn2+‐sensitive dyes. The fractional current carried by Zn2+ (Pf,Zn; 0.5 mM Zn2+ in Ca2+‐ and Mg2+‐free medium) through γ‐ and ε‐AChR channels was 1.7 and 4 %, respectively. 4 P f,Zn increased with the concentration of ZnCl2, but was little affected by physiological concentrations of Ca2+ and Mg2+ in the external medium. 5 The conductance of ACh‐evoked unitary events, measured by cell‐attached or outside‐out recordings, decreased when the patched membrane was exposed to ZnCl2 (1 or 3 mM). Simultaneous application of ACh and Zn2+ to the extra‐patch membrane lengthened channel open duration (τop) by 50%. No obvious increment of τop was observed following exposure of inside‐out patches to Zn2+. 6 The possible physiological relevance of zinc‐induced modulation of AChR channels is discussed.

Hormonal and Neuromuscular Responses to Mechanical Vibration Applied to Upper Extremity Muscles

Objective To investigate the acute residual hormonal and neuromuscular responses exhibited following a single session of mechanical vibration applied to the upper extremities among different acceleration loads. Methods Thirty male students were randomly assigned to a high vibration group (HVG), a low vibration group (LVG), or a control group (CG). A randomized double-blind, controlled-parallel study design was employed. The measurements and interventions were performed at the Laboratory of Biomechanics of the University of LAquila. The HVG and LVG participants were exposed to a series of 20 trials ×10 s of synchronous whole-body vibration (WBV) with a 10-s pause between each trial and a 4-min pause after the first 10 trials. The CG participants assumed an isometric push-up position without WBV. The outcome measures were growth hormone (GH), testosterone, maximal voluntary isometric contraction during bench-press, maximal voluntary isometric contraction during handgrip, and electromyography root-mean-square (EMGrms) muscle activity (pectoralis major [PM], triceps brachii [TB], anterior deltoid [DE], and flexor carpi radialis [FCR]). Results The GH increased significantly over time only in the HVG (Pu200a=u200a0.003). Additionally, the testosterone levels changed significantly over time in the LVG (Pu200a=u200a0.011) and the HVG (Pu200a=u200a0.001). MVC during bench press decreased significantly in the LVG (Pu200a=u200a0.001) and the HVG (Pu200a=u200a0.002). In the HVG, the EMGrms decreased significantly in the TB (Pu200a=u200a0.006) muscle. In the LVG, the EMGrms decreased significantly in the DE (Pu200a=u200a0.009) and FCR (Pu200a=u200a0.006) muscles. Conclusion Synchronous WBV acutely increased GH and testosterone serum concentrations and decreased the MVC and their respective maximal EMGrms activities, which indicated a possible central fatigue effect. Interestingly, only the GH response was dependent on the acceleration with respect to the subjects responsiveness.