Biomedical subjects

Gian Battista Azzena

Publications and source records attributed to Gian Battista Azzena.

11 recordsLinked to original sources

PubMed2006

Motor performance changes induced by muscle vibration.

The possibility that mechanical stimulation of selected muscles can act directly on the nervous system inducing persistent changes of motor performances was explored. On the basis of literature, stimulating parameters were chosen to stimulate the central nervous system and to avoid muscle fibre injuries. A sinusoidal mechanical vibration was applied, for three consecutive days, on the quadriceps muscle in seven subjects that performed a muscular contraction (VC). The same stimulation paradigm was applied on seven subjects in relaxed muscle condition (VR) and seven subjects were not treated at all (NV). Two sessions (PRE and POST) of isometric and isotonic tests were performed separated for 21 days, in all studied groups 7 days before and 15 days after stimulation, whilst an isokinetic test was performed on VC only. In the isometric test, the time of force development showed a significant decrease only in VC (POST vs PRE mean 27.8%, P < 0.05). In the isotonic test, the subjects' had to perform a fatiguing leg extension against a load. In this condition, the fatigue resistance increased greatly in VC (mean 40.3%, P < 0.001), increased slightly in VR and there was no difference in NV. In Isokinetic test, at several angular velocities, significantly less time was required to reach the force peak (mean 20.2% P < 0.05). The findings could be ascribed to plastic changes in proprioceptive processing, leading to an improvement in knee joint control. Such action delineates a new tool in sports training and in motor rehabilitation.

Adaptation, Physiological↗

PubMed2006

Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation.

Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.

Aniline Compounds↗

PubMed2005

Expression of cyclic nucleotide-gated channels in the rat medial vestibular nucleus.

The role of cyclic nucleotide-gated (CNG) channels in sensory signal transduction in retinal and olfactory cells is widely recognized, but there is increasing evidence that they also play more general functions in the central nervous system as downstream effectors of cyclic nucleotides. Here, we demonstrate the expression of the alpha-subunit of rod- and olfactory-type CNG channels (CNG1 and CNG2, respectively) in the rat medial vestibular nucleus (MVN). Nested polymerase chain reaction revealed CNG channel mRNA in the MVN, and CNG1 and CNG2 proteins were also detected by Western blotting and immunohistochemistry. Finally, electrophysiological evidence is provided suggesting that CNG channels play a functional role in the MVN.

Animals↗

PubMed2005

Repeated exposure to pyrrolidine-dithiocarbamate induces peripheral nerve alterations in rats.

Pyrrolidine-dithiocarbamate (PDTC), a synthetic compound widely used in cell biological investigations, recently attracted considerable interest as a putative anticancer agent. However, different dithiocarbamates have previously shown to cause neurological symptoms and morphological alterations in peripheral nerves. The purpose of the present study was to determine whether a 15-day oral administration with low doses of PDTC may produce adverse effects in peripheral nerves of rats. Female Wistar rats were assigned to receive PDTC [0.1, 0.5 or 1.0mmol/(kg body weight/day)] by gavage for 15 days. Reduced conduction velocity was observed by electrophysiological analysis in tibial nerves of treated animals, accompanied by a marked decrease in Shwann cell S100-protein expression determined by immunohistochemistry. Electron microscopy evaluation revealed marked myelin degeneration in the fibers of treated animals. In particular, both morphological and electrophysiological data suggested an impairment of large, fast conducting fibers, whereas the smallest and slowest ones remained intact. However, the activity of plasma and liver alkaline-phosphatase, an enzymic marker of hepatic dithiocarbamate toxicity, was not altered by the treatment. The total contents of the redox-active metal copper increased in tibial nerves of treated rats and was accompanied by raised levels of lipid peroxidation products. This finding suggests a role for oxidative stress in the development of PDTC-induced pathological and functional alterations of tibial nerves. The observation that a 15-day treatment with low doses of PDTC causes functional and morphological derangement of peripheral nerves advices against the possible use of this compound as a chemopreventive agent against cancer.

Alkaline Phosphatase↗

PubMed2005

50-Hz extremely low frequency electromagnetic fields enhance cell proliferation and DNA damage: possible involvement of a redox mechanism.

HL-60 leukemia cells, Rat-1 fibroblasts and WI-38 diploid fibroblasts were exposed for 24-72 h to 0.5-1.0-mT 50-Hz extremely low frequency electromagnetic field (ELF-EMF). This treatment induced a dose-dependent increase in the proliferation rate of all cell types, namely about 30% increase of cell proliferation after 72-h exposure to 1.0 mT. This was accompanied by increased percentage of cells in the S-phase after 12- and 48-h exposure. The ability of ELF-EMF to induce DNA damage was also investigated by measuring DNA strand breaks. A dose-dependent increase in DNA damage was observed in all cell lines, with two peaks occurring at 24 and 72 h. A similar pattern of DNA damage was observed by measuring formation of 8-OHdG adducts. The effects of ELF-EMF on cell proliferation and DNA damage were prevented by pretreatment of cells with an antioxidant like alpha-tocopherol, suggesting that redox reactions were involved. Accordingly, Rat-1 fibroblasts that had been exposed to ELF-EMF for 3 or 24 h exhibited a significant increase in dichlorofluorescein-detectable reactive oxygen species, which was blunted by alpha-tocopherol pretreatment. Cells exposed to ELF-EMF and examined as early as 6 h after treatment initiation also exhibited modifications of NF kappa B-related proteins (p65-p50 and I kappa B alpha), which were suggestive of increased formation of p65-p50 or p65-p65 active forms, a process usually attributed to redox reactions. These results suggest that ELF-EMF influence proliferation and DNA damage in both normal and tumor cells through the action of free radical species. This information may be of value for appraising the pathophysiologic consequences of an exposure to ELF-EMF.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2004

Electrophysiological and molecular evidence of L-(Cav1), N- (Cav2.2), and R- (Cav2.3) type Ca2+ channels in rat cortical astrocytes.

Changes in intracellular Ca2+ levels are an important signal underlying neuron-glia cross-talk, but little is known about the possible role of voltage-gated Ca2+ channels (VGCCs) in controlling glial cell Ca2+ influx. We investigated the pharmacological and biophysical features of VGCCs in cultured rat cortical astrocytes. In whole-cell patch-clamp experiments, L-channel blockade (5 microM nifedipine) reduced Ba2+ current amplitude by 28% of controls, and further decrease (32%) was produced by N-channel blockade (3 microM omega-conotoxin-GVIA). No significant additional changes were observed after P/Q channel blockade (3 microM omega-conotoxin-MVIIC). Residual current (36% of controls) amounted to roughly the same percentage (34%) that was abolished by R-channel blockade (100 nM SNX-482). Electrophysiological evidence of L-, N-, and R-channels was associated with RT-PCR detection of mRNA transcripts for VGCC subunits alpha1C (L-type), alpha1B (N-type), and alpha1E (R-type). In cell-attached recordings, single-channel properties (L-currents: amplitude, -1.21 +/- 0.02 pA at 10 mV; slope conductance, 22.0 +/- 1.1 pS; mean open time, 5.95 +/- 0.24 ms; N-currents: amplitude, -1.09 +/- 0.02 pA at 10 mV; slope conductance, 18.0 +/- 1.1 pS; mean open time, 1.14 +/- 0.02 ms; R-currents: amplitude, -0.81 +/- 0.01 pA at 20 mV; slope conductance, 10.5 +/- 0.3 pS; mean open time, 0.88 +/- 0.02 ms) resembled those of corresponding VGCCs in neurons. These novel findings indicate that VGCC expression by cortical astrocytes may be more varied than previously thought, suggesting that these channels may indeed play substantial roles in the regulation of astrocyte Ca2+ influx, which influences neuron-glia cross-talk and numerous other calcium-mediated glial-cell functions.

Animals↗

PubMed2004

Functional activity and expression of inducible nitric oxide synthase (iNOS) in muscle of the isolated distal colon of mdx mice.

The inducible isoform of nitric oxide (NO) synthase (iNOS), expressed in endothelium, epithelium, and inflammatory cells, produces a large amount of NO. Previous studies on mouse intestine indicate that a muscular iNOS may have a role in the storage of intraluminal content. In this study we investigated the presence and function of iNOS in the colonic smooth muscle cells of 2- and 12-month-old dystrophic (mdx) mice. By using an in vitro isovolumic technique, and immunohistochemical and Western blot analysis, we demonstrated that iNOS is expressed and active in the smooth muscle cells of normal mouse and defective in young adult (2-month-old) mdx mice. Therefore, an altered activity of the muscle iNOS might explain the motility disorders observed in the colon of mdx mice and, from a clinical point of view, the impairment of intestinal function in dystrophic patients.

Animals↗

PubMed2004

Effects of 50 Hz electromagnetic fields on voltage-gated Ca2+ channels and their role in modulation of neuroendocrine cell proliferation and death.

Possible correlation between the effects of electromagnetic fields (EFs) on voltage-gated Ca(2+) channels, cell proliferation and apoptosis was investigated in neural and neuroendocrine cells. Exposure to 50 Hz EFs significantly enhanced proliferation in human neuroblastoma IMR32 (+40%) and rat pituitary GH3 cells (+38%). In IMR32 cells EF stimulation also inhibited puromycin- and H(2)O(2)-induced apoptosis (-22 and -33%, respectively). EF effects on proliferation and apoptosis were counteracted by Ca(2+) channel blockade. In whole-cell patch-clamp experiments 24-72 h exposure to EFs increased macroscopic Ba(2+)-current density in both GH3 (+67%) and IMR32 cells (+40%). Single-channel recordings showed that gating of L and N channels was instead unaffected, thus suggesting that the observed enhancement of current density was due to increased number of voltage-gated Ca(2+) channels. Western blot analysis of plasma membrane-enriched microsomal fractions of GH3 and IMR32 cells confirmed enhanced expression of Ca(2+) channel subunit alpha(1) following exposure to EFs. These data provide the first direct evidence that EFs enhance the expression of voltage-gated Ca(2+) channels on plasma membrane of the exposed cells. The consequent increase in Ca(2+) influx is likely responsible for the EF-induced modulation of neuronal cell proliferation and apoptosis.

Animals↗

PubMed2004

Modulation of Ca(v)1 and Ca(v)2.2 channels induced by nitric oxide via cGMP-dependent protein kinase.

The unconventional gaseous transmitter nitric oxide (NO) markedly influences most of mechanisms involved in the regulation of intracellular Ca2+ homeostasis. In excitable cells, Ca2+ signaling mainly depends on the activity of voltage-gated Ca2+ channels (VGCCs). In the present paper, we will review data from our laboratory and others characterizing NO-induced modulation of Ca(v)1 (L-type) and Ca(v)2.2 (N-type) channels. In particular, we will explore experimental evidence indicating that NO's inhibition of channel gating is produced via cGMP-dependent protein kinase and examine some of the numerous cell functions that are potentially influenced by the action of NO on Ca2+ channels.

Animals↗

PubMed2004

Nitric oxide increases the spontaneous firing rate of rat medial vestibular nucleus neurons in vitro via a cyclic GMP-mediated PKG-independent mechanism.

The effects of nitric oxide (NO) on the discharge rate of medial vestibular nucleus neurons (MVNn) were investigated in rat brainstem slices. The NO-donor sodium nitroprusside (SNP, 200 microM) caused a marked enhancement (+36.7%) of MVNn spontaneous firing rate, which was prevented by the NO-scavenger, carboxy-PTIO (300 microM). The SNP effects were not modified (+37.4%) by synaptic uncoupling, suggesting that NO influences intrinsic membrane properties of MVNn rather than the synaptic input they receive. The excitatory action of SNP was virtually abolished by slice pretreatment with the soluble guanylyl cyclase inhibitor, ODQ (10 microM), and it was mimicked (+33.1%) by the cGMP analogue 8-Br-cGMP (400 microM). Protein kinase G (PKG) and cAMP/protein kinase A (PKA) were both excluded as downstream effectors of the NO/cGMP-induced excitation. However, the cyclic nucleotide-gated (CNG) channel blockers, L-cis-diltiazem (LCD, 100 microM) and Sp-8-Br-PET-cGMPS (100 microM), significantly reduced the firing rate increase produced by 8-Br-cGMP. Moreover, LCD alone decreased spontaneous MVNn firing (-19.7%), suggesting that putative CNG channels may contribute to the tonic control of resting MVNn discharge. 8-Br-cAMP (1 mM) also elicited excitatory effects in MVNn (+40.8%), which occluded those induced by 8-Br-cGMP, indicating that the two nucleotides share a common target. Finally, nested-polymerase chain reaction assay revealed the expression of CNG channel alpha subunit transcript in MVNn. Our data provide the first demonstration that NO/cGMP signalling modulates MVNn spontaneous firing through a mechanism that is independent of PKG or PKA and probably involves activation of CNG channels.

Action Potentials↗

PubMed2002

cGMP/protein kinase G-dependent inhibition of N-type Ca2+ channels induced by nitric oxide in human neuroblastoma IMR32 cells.

Although data from our laboratory and others suggest that nitric oxide (NO) exerts an overall inhibitory action on high-voltage-activated Ca2+ channels, conflicting observations have been reported regarding its effects on N-type channels. We performed whole-cell and cell-attached patch-clamp recordings in IMR32 cells to clarify the functional role of NO in the modulation of N channels of human neuronal cells. During depolarizing steps to +10 mV from V(h) = -90 mV, the NO donor, sodium nitroprusside (SNP; 200 microm), reduced macroscopic N currents by 34% (p < 0.01). The magnitude of inhibition was similar at all voltages tested (range, -40 to +50 mV). No significant inhibition was observed when SNP was applied together with the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium salt (300 microm), or after cell treatment with the guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one (10 microm). 8-bromoguanosine-cGMP (8-Br-cGMP) (400 microm) mimicked the effects of SNP, reducing Ba2+ currents by 37% (p < 0.001). Cell treatment with the protein kinase G (PKG) inhibitor KT5823 (1 microm) or guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chloro-phenylthio)-Rp-isomer, triethylammonium salt (20 microm) virtually abolished the effects of 8-Br-cGMP. At the single-channel level, 8-Br-cGMP reduced the channel open probability by 59% and increased both the mean shut time and the null sweep probability, but it had no significant effects on channel conductance, mean open time, or latency of first openings. These data suggest that NO inhibits N-channel gating through cGMP and PKG. The consequent decrease in Ca2+ influx through these channels may affect different neuronal functions, including neurotransmitter release.

Calcium↗