Comparison of the use of gel threads and microcarrier beads in Li+ transport studies of human neuroblastoma SH-SY5Y cells

Nikolakopoulos, J.; Zachariah, Cherian; Freitas, Duarte Mota de; Geraldes, Carlos F. G. C.

doi:10.1016/s0020-1693(96)05273-5

Cited by 9 publications

(10 citation statements)

References 15 publications

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“…One obstacle to the use of this cell line for fluorescence studies was that the cells are very delicate. For NMR experiments, the cells are embedded in gel threads or there is some other means of attachment, and the cells are perfused with medium (Nikolakopoulos et al, 1996(Nikolakopoulos et al, , 1998Zachariah et al, 1996). However, this was not practical with our fluorescence setup.…”

Section: Discussionmentioning

confidence: 99%

“…This cell line has also been used in an investigation of the ion regulatory properties of Li ϩ and other metal ions such as Ca 2ϩ (McDonald et al, 1994). Unlike primary cultures of neurons and glial cells, the SH-SY5Y cell line provides significantly higher cell densities, which are necessary for Li NMR (Nikolakopoulos et al, 1996) and fluorescence measurements. Thus far, we have used the SH-SY5Y cell line in our laboratory to study the transport and binding properties of Li ϩ by 7 Li NMR spectroscopy (Nikolakopoulos et al, 1996(Nikolakopoulos et al, , 1998Zachariah et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

“…To perform perfused NMR experiments on the SH-SY5Y cell line, we had to immobilize the cells by using a technique that involved entrapment of the cells in agarose threads. The agarose threads containing the SH-SY5Y cells were perfused with fresh medium throughout the course of the experiment, which enabled the cells to maintain a high degree of viability (Nikolakopoulos et al, 1996(Nikolakopoulos et al, , 1998Egan, 1987;Swergold, 1992).…”

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confidence: 99%

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Competition between Li+ and Mg2+ in Neuroblastoma SH-SY5Y Cells: A Fluorescence and 31P NMR Study

Amari

Layden

Nikolakopoulos

et al. 1999

Biophysical Journal

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Because Mg2+ and Li+ ions have similar chemical properties, we have hypothesized that Li+/Mg2+ competition for Mg2+ binding sites is the molecular basis for the therapeutic action of lithium in manic-depressive illness. By fluorescence spectroscopy with furaptra-loaded cells, the free intracellular Mg2+ concentration within the intact neuroblastoma cells was found to increase from 0. 39 +/- 0.04 mM to 0.60 +/- 0.04 mM during a 40-min Li+ incubation in which the total intracellular Li+ concentration increased from 0 to 5.5 mM. Our fluorescence microscopy observations of Li+-free and Li+-loaded cells also indicate an increase in free Mg2+ concentration upon Li+ incubation. By 31P NMR, the free intracellular Mg2+ concentrations for Li+-free cells was 0.35 +/- 0. 03 mM and 0.80 +/- 0.04 mM for Li+-loaded cells (final total intracellular Li+ concentration of 16 mM). If a Li+/Mg2+ competition mechanism is present in neuroblastoma cells, an increase in the total intracellular Li+ concentration is expected to result in an increase in the free intracellular Mg2+ concentration, because Li+ displaces Mg2+ from its binding sites within the nerve cell. The fluorescence spectroscopy, fluorescence microscopy, and 31P NMR spectroscopy studies presented here have shown this to be the case.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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Competition between Li+ and Mg2+ in Neuroblastoma SH-SY5Y Cells: A Fluorescence and 31P NMR Study

Amari

Layden

Nikolakopoulos

et al. 1999

Biophysical Journal

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“…(2) yields an apparent k i value, k iapp , because for the cells immobilised in agarose gel threads the influx rate constant has a contribution from the diffusion process of Li + across the gel before reaching the cell membrane [44]. The average value obtained for k iapp is 0.012 F 0.003 min À 1 .…”

Section: + Influx and Intracellular Degree Of Immobilisation Bymentioning

confidence: 99%

Effects of Li+ transport and intracellular binding on Li+/Mg2+ competition in bovine chromaffin cells

Fonseca

Montezinho

Nabais

et al. 2004

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Li(+) transport, intracellular immobilisation and Li(+)/Mg(2+) competition were studied in Li(+)-loaded bovine chromaffin cells. Li(+) influx rate constants, k(i), obtained by atomic absorption (AA) spectrophotometry, in control (without and with ouabain) and depolarising (without and with nitrendipine) conditions, showed that L-type voltage-sensitive Ca(2+) channels have an important role in Li(+) uptake under depolarising conditions. The Li(+) influx apparent rate constant, k(iapp), determined under control conditions by (7)Li NMR spectroscopy with the cells immobilised and perfused, was much lower than the AA-determined value for the cells in suspension. Loading of cell suspensions with 15 mmol l(-1) LiCl led, within 90 min, to a AA-measured total intracellular Li(+) concentration, [Li(+)](iT)=11.39+/-0.56 mmol (l cells)(-1), very close to the steady state value. The intracellular Li(+) T(1)/T(2) ratio of (7)Li NMR relaxation times of the Li(+)-loaded cells reflected a high degree of Li(+) immobilisation in bovine chromaffin cells, similar to neuroblastoma, but larger than for lymphoblastoma and erythrocyte cells. A 52% increase in the intracellular free Mg(2+) concentration, Delta[Mg(2+)](f)=0.27+/-0.05 mmol (l cells)(-1) was measured for chromaffin cells loaded with the Mg(2+)-specific fluorescent probe furaptra, after 90-min loading with 15 mmol l(-1) LiCl, using fluorescence spectroscopy, indicating significant displacement of Mg(2+) by Li(+) from its intracellular binding sites. Comparison with other cell types showed that the extent of intracellular Li(+)/Mg(2+) competition at the same Li(+) loading level depends on intracellular Li(+) transport and immobilisation in a cell-specific manner, being maximal for neuroblastoma cells.

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“…It should be noticed that the k i values determined for the immobilised cells are in fact apparent k i (k iapp ) because of the contribution of the diffusion process of Li ? across the gel before reaching the cells (Fonseca et al 2004;Nikolakopoulos et al 1996). The k iapp value obtained in this study for chromaffin cells immobilised in agarose gel threads (k iapp = 0.0108 ± 0.0004 min -1 or k = 1.54 ± 0.06 h, at [Li ? ]…”

Section: Discussionmentioning

confidence: 82%

23Na multiple quantum filtered NMR characterisation of Na+ binding and dynamics in animal cells: a comparative study and effect of Na+/Li+ competition

Fonseca

Montezinho

et al. 2013

Eur Biophys J

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Double quantum and triple quantum filtered (23)Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na(+) in different cellular systems, in the absence and presence of Li(+). The kinetics of Li(+) influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li(+) than red blood cells (RBCs), while a higher intracellular Li(+) concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na(+) in all cellular systems studied. Isotropic intracellular Na(+) motions were observed in all types of cells, while anisotropic Na(+) motions in the intracellular compartment were only detected in RBCs. (23)Na triple quantum signal efficiency for intracellular Na(+) was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. (23)Na double quantum signal efficiency for intracellular Na(+) was non-zero only in RBCs, and for extracellular Na(+) the order RBCs > chromaffin > SH-SY5Y cells was observed. Li(+) loading generally decreased intracellular Na(+) isotropic movements in the cells, except for astrocytes incubated with a low Li(+) concentration and increased anisotropic intracellular Na(+) movements in RBCs. Li(+) effects on the extracellular signals were more complex, reflecting Li(+)/Na(+) competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li(+) action.

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Comparison of the use of gel threads and microcarrier beads in Li+ transport studies of human neuroblastoma SH-SY5Y cells

Cited by 9 publications

References 15 publications

Competition between Li+ and Mg2+ in Neuroblastoma SH-SY5Y Cells: A Fluorescence and 31P NMR Study

Competition between Li+ and Mg2+ in Neuroblastoma SH-SY5Y Cells: A Fluorescence and 31P NMR Study

Effects of Li+ transport and intracellular binding on Li+/Mg2+ competition in bovine chromaffin cells

23Na multiple quantum filtered NMR characterisation of Na+ binding and dynamics in animal cells: a comparative study and effect of Na+/Li+ competition

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