Biomedical subjects

Carla Marchetti

Publications and source records attributed to Carla Marchetti.

9 recordsLinked to original sources

PubMed2006

Nickel differentially affects NMDA receptor channels in developing cultured rat neurons.

Nickel (Ni(2+)) is a transition metal that exerts multiple and complex effects on N-methyl-d-aspartate (NMDA) channels. In both HEK293 cells and Xenopus laevis oocytes expressing recombinant NMDA receptors, Ni(2+) (<100 microM) caused a potentiation of NR2B-containing channels but a voltage-independent inhibition in those containing NR2A. We took advantage of this different response to investigate the developmental switch between NR2B and NR2A subunits in neonatal rat cerebellar granule cells up to 16 days in vitro (DIV) and in rat embryo cortical neurons up to 35 DIV. In both cultures, the effect of Ni(2+) on the NMDA current gradually changed from potentiating to inhibitory with progressing DIV, and the decline of potentiation correlated well with the decrease in sensitivity for the NR2B specific antagonist ifenprodil. Dose-dependent experiments confirmed that Ni(2+) has a different effect in younger cultures with respect to older ones, in agreement with an increase of the percentage of NR2A-containing receptors. The developmental switch occurred within the first 5 DIV in cerebellar granule cells and after 20 DIV in cortical neurons. All these data indicate that Ni(2+) is a suitable marker for the identification of NR2A and NR2B native channel subunits and can be used to trace the development of NMDA receptor composition.

Analysis of Variance↗

PubMed2005

NMDA receptors as targets of heavy metal interaction and toxicity.

The N-methyl-D-aspartate (NMDA) receptor (NR) is a ligand-gated channel that carries the slow component of the glutamate-activated postsynaptic current. Divalent metal ions can affect the NR channel activity in a voltage-dependent [Mg(II)-like] or voltage-independent [Zn(II)-like] manner. We have studied the effect of two toxic metals, lead [Pb(II)] and nickel [Ni(II)] on recombinant NR1a-NR2A and NR1a-NR2B channels expressed in RNA-injected Xenopus laevis oocytes or in transiently transfected mammalian HEK293 cells. Pb(II) caused a dose-dependent, but voltage-independent reversible inhibition of NMDA-activated channel activity similar for NR2A and NR2B-containing receptors; it did not modify the single channel conductance, indicating that its binding site is located out of the ionic pathway of permeation. On the contrary, Ni(II) had multiple and complex effects on NR channels. It determined a voltage-dependent, Mg(II)-like block by which the single channel amplitude and the mean open time were reduced in both NR2A and NR2B-containing channels. While high (>100 microM) concentrations caused a dose-dependent reduction of the activity in both channel types, 30 microM determined a voltage-independent decrease in the frequency of NR1a-NR2A channel openings, but an increase in the frequency of NR1a-NR2B channel openings, confirming previous observations of a subunit-dependent effect of this metal. These results were interpreted under the hypothesis that Pb(II) mediates a Zn(II)-like voltage-independent allosteric modulation that, different from Zn(II), is subunit-independent. In contrast, Ni(II) has different modes of action, which are dependent on the NR2 subunit type present in the receptor and are likely to be related to different interaction sites. The NR2B-dependent facilitation bears close similarities with the polyamine-mediated potentiation.

Animals↗

PubMed2005

Susceptibility of insulinoma cells to cadmium and modulation by L-type calcium channels.

Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulates in pancreas and may be a cause of diabetes in humans. In the insulinoma cells line HIT-T15 (HIT), we measured internal calcium (Ca) and Cd levels by the fluorescent dye Fura-2 and confirm that L-type voltage-dependent calcium channels (VDCC) play a major role in glucose response and represent a pathway of Cd influx in these cells. Therefore we examined the role of VDCC in acute Cd poisoning by comparing its accumulation and cytotoxic effect in HIT cells and in epithelial-like VDCC-free HeLa cells. Cultures were incubated with 10-300 microM Cd for 15 min-6 h. While negligible at the end of the treatment, HIT cell death was evident after 18-24 h, and it was time-, dose- and serum-dependent. Short (< or = 60 min) Cd treatments with lower doses (< or = 100 microM in serum-free medium) induced delayed apoptotic cell death, as demonstrated by DNA fragmentation on agarose gels and segmentation of DAPI-stained nuclei. Longer incubations and/or higher concentrations caused mainly necrosis. The same treatments were largely harmless in HeLa cells, in which neither death nor DNA fragmentation was observed. The Ca antagonist nimodipine was capable to prevent HIT cell death at lower doses of Cd and to restore the apoptotic condition at higher doses, indicating that reduction of Cd flux through VDCC modulates Cd toxicity. These data demonstrate a specific sensitivity to Cd of insulinoma cells that can be significant for pancreatic beta-cell pathology.

Animals↗

PubMed2005

Two-photon analysis of lead accumulation in rat cerebellar granule neurons.

Lead (Pb2+) is a common pollutant and potent central neurotoxin. We have studied its pathways of permeation by two-photon fluorescence microscopy in rat cerebellar granule neurons loaded with the fluorescent dye indo-1. Pb2+ binds indo-1 with high affinity acting as a quencher. Its permeation through the neuronal membrane was indicated by a decrease of the fluorescence emission, which occurred even in resting condition. In the presence of 20 microM Pb2+, uptake reached a plateau level (approximately 45% of initial fluorescence) in 4 min and was partially antagonized by 25 microM lanthanum. Subsequent addition of a membrane permeant ionophore caused a further (>70%) quenching of the dye, suggesting that previous saturation was due to inactivation of the transport system. Intracellular Pb2+ concentrations were evaluated from the fluorescence intensity and this estimate indicated that the concentration of free Pb2+ sufficient to inactivate the transport system is close to 50 pM.

Animals↗

PubMed2004

Two-photon imaging of calcium accumulation in rat cerebellar granule cells.

Topical accumulation of calcium ions in neurites and cell bodies of rat cerebellar granule cells was studied by two-photon microscopy in neurons loaded with the Ca-sensitive fluorescent indicator Oregon Green 488 Bapta. High potassium caused a rapid surge of internal calcium ([Ca2+]i) in the cell body, followed by a plateau. In neurites, [Ca2+]i reached a peak and then decreased back to the control level. In contrast, in neurons stimulated by NMDA, [Ca2+]i reached a steady level and remained constant as long as the agonist was present in the bath, either in the cell bodies or in neurites. In the latter, the response to NMDA treatment was smaller and heterogeneous, and [Ca2+]i increased in certain segments of the neurite, but not in others.

Animals↗

PubMed2003

Subunit-dependent effects of nickel on NMDA receptor channels.

Nickel (Ni2+) is a transition metal that affects different neuronal ionic channels. We investigated its effects on glutamate channels of the NMDA-type in the presence of saturating concentration of glutamate or NMDA (50 microM), in 0 external Mg and in the continuous presence of saturating glycine (30 microM). In neonatal rat cerebellar granule cells, Ni2+ inhibited the current evoked by NMDA at -60 mV with an IC50 close to 40 microM. The inhibition was weakly voltage-dependent and the current at +40 mV was inhibited with IC50=86 microM. Wash out of the metal unmasked a stimulatory effect which persisted for a few seconds. In HEK293 cells transiently transfected with recombinant NR1a-NR2A receptors, Ni2+ inhibited the current elicited by glutamate with an IC50=52 microM at -60 mV and 90 microM at +40 mV. In HEK293 expressing NR1a-NR2B receptors, 0.1-100 microM Ni2+ caused a potentiation of the current, with EC50=4 microM, while with 300 microM, a voltage-dependent block became apparent (IC50=170 microM). As previously reported, the current through both classes of recombinant receptors was steeply dependent on external pH, and in both cases the protonic block had an IC50 close to pH 7.2. Application of Ni2+ showed that stimulation of NR1a-NR2B receptor channels was dependent on external pH, while voltage-independent inhibition of NR1a-NR2A was less sensitive to pH change. These results indicate that Ni2+ has multiple and complex effects on NMDA channels, which are largely dependent on the NR2 subunit.

Animals↗

PubMed2003

Molecular targets of lead in brain neurotoxicity.

The detrimental effects of lead poisoning have been well known since ancient times, but some of the most severe consequences of exposure to this metal have only been described recently. Lead [Pb(II)] affects the higher functions of the central nervous system and undermines brain growth, preventing the correct development of cognitive and behavioral functions. As an established neurotoxin, Pb(II) crosses the blood-brain barrier rapidly and concentrates in the brain. The mechanisms of lead neurotoxicity are complex and still not fully understood, but recent findings recognized that both Ca(II) dependent proteins and neurotransmitters receptors represent significant targets for Pb(II). In particular, acute and chronic exposure to lead would predominantly affect two specific protein complexes: protein kinase C and the N-methyl-D-aspartate subtype of glutamate receptor. These protein complexes are deeply involved in learning and cognitive functions and are also thought to interact significantly with each other to mediate these functions. This review outlines the most recent hypotheses and evidences that link lead poisoning to impairment of these protein functions, as well as the in vitro experimental approaches that are most likely to provide information on basic mechanicistic processes.

Animals↗

PubMed2002

Lymphatic vessels in the oral cavity: different structures for the same function.

A study using a light and transmission electron microscope was performed on some structural characteristics of the lymphatic capillaries in different regions of the human oral cavity. The lymphatic capillaries of dental pulp, masticatory mucosa (gingiva and peri-implant mucosa) and lining mucosa (cheek) were examined. Our attention was focused on the morphologic characteristics of the endothelial wall in the lymphatic capillaries. In particular, the connections between endothelial cells were investigated. In the lymphatic capillaries of the dental pulp, the endothelial wall was always very complex. It frequently presented protrusions of the endothelial cells that overlapped and formed intercellular channels. These channels were thus contained by the vessel endothelial wall with their extremities opening out towards the surrounding interstitium and the vessel lumen. The endothelial wall of the lymphatic capillaries of the cheek was very smooth and thin without complex intercellular junctions. The endothelial cells were joined by end-to-end junctions and open junctions were frequently observed. Intercellular channels were also found in the endothelial wall of lymphatic capillaries of the gingiva and the peri-implant mucosa. The presence of numerous clefts represented by the open junctions in the lymphatics of the cheek and the existence of complex intercellular adhesions with the formation of intercellular channels in the endothelial wall of the lymphatic capillaries of the dental pulp and gingiva induce us to believe that these may play a role in the various mechanisms used by lymphatic capillaries to absorb interstitial fluids. These mechanisms are based on the different morpho-functional characteristics of the surrounding tissue.

Capillaries↗

PubMed2002

Microscopic, immunocytochemical, and ultrastructural properties of peri-implant mucosa in humans.

BACKGROUND: Microscopic and immunocytochemical studies have demonstrated both similarities and differences between the gingival mucosa and the peri-implant mucosa restored around dental implants. METHODS: This study was performed on 10 samples of peri-implant mucosa from 10 patients who had undergone implant treatment 16 to 18 months before. Microscopic, ultrastructural, and immunocytochemical investigations were performed to characterize the epithelial and connective layers of the peri-implant mucosa. RESULTS: All specimens showed the morphologic characteristics of well restored tissues. The immunocytochemical reactions used to detect cytokeratins demonstrated that the restored peri-implant mucosa had a non-keratinized epithelium. The apical part of the inner epithelium was a few cell layers thick, like the corresponding junctional epithelium, but it stained positively with the markers for the cytokeratins expressed by the stratified epithelia. Ultrastructurally the keratinocytes adjacent to the implant displayed nuclei containing a rich cytoplasm with filaments and specialized intercellular junctions right up to the more superficial strata. Immunocytochemical reactions and ultrastructural observations demonstrated the presence of non-keratinocyte cells; i.e., Langerhans cells, melanocytes, and Merkel cells. Several cells were found to be proliferating by immunoreaction with mAb anti-PCNA, while immunoreactions with mAbs to detect von Willebrand factor, CD34, and vascular endothelial growth factor marked the well-developed networks of blood and lymphatic vessels in the connective tissue. S-100 and protein gene product 9.5 positive nerve fibers were marked. Immunocytochemical reactions with mAbs anti-vimentin, anti-laminin were also performed. CONCLUSIONS: Our results demonstrated that all the epithelial and connective components of the mucosa are involved in the substantial regrowth of the peri-implant tissue and subsequently in the success of the implant.

Actin Cytoskeleton↗