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Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-α, interleukin-1-β, and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A1 receptors (A1R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of adenosine from microglia. In this study, we show that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 as reducing adenosine levels in hippocampal cultures, by adenosine deaminase treatment, strongly impairs CX3CL1-mediated neuroprotection. Furthermore, we confirm the predominant role of microglia in mediating the neuronal effects of CX3CL1, because the selective depletion of microglia from hippocampal cultures treated with clodronate-filled liposomes causes the complete loss of effect of CX3CL1. We also show that hippocampal neurons obtained from A1R−/− mice are not protected by CX3CL1 whereas A2AR−/− neurons are. The requirement of functional A1R for neuroprotection is not unique for CX3CL1 as A1R−/− hippocampal neurons are not rescued from Glu-induced cell death by other neurotrophins such as brain-derived neurotrophic factor and erythropoietin, which are fully active on wt neurons. [ABSTRACT FROM AUTHOR]

Breath-by-breath O(2) uptake (VO2, L min(-1)) and blood lactate concentration were measured before, during exercise, and recovery in six kata and six kumite karate Word Champions performing a simulated competition. VO2max, maximal anaerobic alactic, and lactic power were also assessed. The total energy cost (VO2TOT mL kg(-1) above resting) of each simulated competition was calculated and subdivided into aerobic, lactic, and alactic fractions. Results showed that (a) no differences between kata and kumite groups in VO2max, height of vertical jump, and Wingate test were found; (b) VO2TOT were 87.8 +/- 6.6 and 82.3 +/- 12.3 mL kg(-1) in kata male and female with a performance time of 138 +/- 4 and 158 +/- 14 s, respectively; 189.0 +/- 14.6 mL kg(-1) in kumite male and 155.8 +/- 38.4 mL kg(-1) in kumite female with a predetermined performance time of 240 +/- 0 and 180 +/- 0 s, respectively; (c) the metabolic power was significantly higher in kumite than in kata athletes (p < or = 0.05 in both gender); (d) aerobic and anaerobic alactic sources, in percentage of the total, were significantly different between gender and disciplines (p < 0.05), while the lactic source was similar; (e) HR ranged between 174 and 187 b min(-1) during simulated competition. In conclusion, kumite appears to require a much higher metabolic power than kata, being the energy source with the aerobic contribution predominant. [ABSTRACT FROM AUTHOR]

Patch-clamp recordings were used to study the activation of ion channels in the cell membrane of cultured embryonic chick myotubes treated with the specific activator of protein kinase C, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; 1 × 10 M). Myotubes exhibited a spontaneous channel activity when the TPA-induced dedifferentiative processes developed. This consisted in the activation of inward current channels (∼ 35 pS conductance; ∼ 6 ms open time). These spontaneously active channels were insensitive to α-bungarotoxin, curare, atropine and tetrodotoxin and were not inhibited by the withdrawal of TPA. It is suggested that a prolonged stimulation of the protein kinase C causes a irreversible deregulation of the membrane channel function during cell dedifferentiation. [ABSTRACT FROM AUTHOR]

The effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of protein kinase C (PKc), were examined on the frog neuromuscular junction. The depolarization elicited by iontophoretically applied acetylcholine (ACh) was reversibly decreased by 20-60% when muscle fibres were exposed to 1-5×10 M TPA. Liposome-delivered phosphatidylcholine (100 μg/ml) prevented this effect. A similar decrease in ACh-sensitivity was produced by diacylglycerol (diolein), a physiological activator of PKc, but in this case the decrease was only partially reversible. In TPA-Ringer, (1) the peak size of miniature end-plate potentials exhibited a small decrease; (2) miniature end-plate currents were reduced in size and their decay time constant became longer and relatively independent of membrane potential. The possibility that these TPA-induced actions are mediated by activation of PKc is discussed. [ABSTRACT FROM AUTHOR]

The rate of desensitization of nicotinic acetylcholine (ACh) receptor (nAChR), an important characteristic of nAChR function, was studied in myotubes of the mouse C2C12 cell line at different times after fusion, by measuring the decay of ACh-evoked currents ( I) under various patch-clamp configurations. We observed a progressive slowing of I decay rate (half-decay time rose from about 0.5 s to over 5 s) in myotubes of increasing size (i.e. age) under all experimental conditions, except in outside-out patches, when I decayed as fast as in the smallest myotubes. Single-channel conductance (about 35 pS) and open time (about 3.5 ms), measured in outside-out and cell-attached patches, were independent of myotube size. In Xenopus oocytes injected with poly(A)RNA extracted from C2C12 myoblasts or mature myotubes, I decay was about 50 times slower than in myotubes. Neither cAMP-dependent nor diacylglycerol-dependent protein kinases, actin nor microtubule polymerization state influenced I decay. Our data indicate that the cellular environment, but not readily dialysable cytosolic factors, markedly influences the functional behaviour of nAChR. [ABSTRACT FROM AUTHOR]

In mouse C2C12 myotubes acetylcholine (ACh) elevates the concentration of myoplasmic Ca ([Ca]) by inducing Ca influx through transmittergated and voltage-gated channels, and by mobilizing Ca from internal stores. The relative contribution of each of these ACh-activated sources to the global [Ca] elevation was estimated. We found that Ca entry through voltage- and ACh-gated channels accounts for roughly 80% of the total [Ca] increment, while mobilization from internal caffeine-sensitive and inositol-trisphosphate (Ins P) sensitive stores contributes the remaining 20% to the maximal [Ca] increment. Furthermore, we found that ACh-induced mobilization from Ins P-sensitive stores also develops in embryonic chick myotubes. The differential importance of the Ca signalling pathways activated by ACh during myogenesis is discussed. [ABSTRACT FROM AUTHOR]

The cholinergic responses of the human tumour cell line TE671/RD were examined using digital Ca imaging fluorescence microscopy and patch-clamp measurements. In response to stimulation of the muscarinic acetylcholine (ACh) receptor (mAChR), the intracellular concentration of Ca ([Ca]) rose about two-fold, in parallel with inositol 1,4,5-trisphosphate accumulation, measured by chromatographic techniques. By contrast, there was no increment of [Ca] upon stimulation of the nicotinic ACh receptor (nAChR), nor after caffeine application. Electrophysiological experiments showed that TE671/RD cells lack functional voltage-activated Ca channels. The stimulation of the nAChR induced transient whole-cell currents ( I). Little or no current was detected in isotonic extracellular Ca, with Cs in the patch pipette. Cell pretreatment with muscarine reduced I by about 20%, without consistent modifications of current kinetics. Muscarine applied to the extra-patch membrane under the cell-attached configuration had no obvious effect on ACh-evoked unitary events. In conclusion, in human TE671/ RD cells, muscarinic stimulation increases [Ca], while nicotinic stimulation does not. In addition, the nAChR exhibits peculiar ion permeability properties and is not functionally regulated by the breakdown of phosphoinositides. [ABSTRACT FROM AUTHOR]

The effect of the antiviral agent interferon (IFN) on the function of the nicotinic acetylcholine receptor (AChR) channel, has been investigated in both mammalian cultured myotubes and adult fibres, using the single channel recording patch-clamp technique. Shortened AChR-channel lifetime, and occasionally reduced channel conductance and slowed opening frequency were seen with fibroblast IFN (IFN- β) in the mouse myotubes, and with IFN- β and leucocytes IFN (IFN-α), in the rat muscle fibres. These effects paralleled an increase in the cytosolic level of cAMP. This suggests that IFN exerts a regulatory action on AChR function. A similar regulatory action on other receptor may be responsible for some of the neurological side effects observed in patients treated with IFN. [ABSTRACT FROM AUTHOR]

Junctional transfer of ions in monolayer primary cultures of Sertoli cells has been studied by using conventional techniques. Cells were extensively electrically coupled. Membrane voltage displacements caused by the injected current pulses were measured at variable interelectrode distance. Data analyses were based on a thin-sheet model for current flow )Jongsma and van Tijn, 1972) and yielded a generalized space constant of 248 um and an internal resistivity of 1.7 k0hm.cm in standard medium. Electrical coupling was inhibited by A23187 Ca-ionophore (5 uM). [ABSTRACT FROM AUTHOR]