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Calcium-mediated transductive systems and functionally active gap junctions in astrocyte-like GL15 cells.

Mariggio MA, Mazzoleni G, Pietrangelo T, Guarnieri S, Morabito C, Steimberg N, Fano G - BMC Physiol. (2001)

Bottom Line: In particular, oscillations in intracellular Ca2+ levels were recorded either spontaneously, or in the presence of ATP or glutamate (but not KCl).The protein is organised in characteristic spots on the plasma membrane at cell-cell contact regions, and its presence and distribution depends on the differentiative status of the cell.In conclusion, results from this study support the use of the GL15 cell line as a suitable in vitro astrocyte model, which provides a valuable guide for studying glial physiological features at various differentiation phases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Scienze del Farmaco, Laboratorio di Fisiologia Cellulare, Università G, D'Annunzio, I-66013 Chieti. mariggio@unich.it

ABSTRACT

Background: It has been proposed that GL15, a human cell line derived from glioblastoma multiforme, is a possible astroglial-like cell model, based on the presence of cytoplasmic glial fibrillary acidic protein.

Results: The aim of this work was to delineate the functional characteristics of GL15 cells using various experimental approaches, including the study of morphology, mechanism of induction of intracellular Ca2+ increase by different physiological agonists, and the presence and permeability of the gap-junction system during cell differentiation. Immunostaining experiments showed the presence and localization of specific glial markers, such as glial fibrillary acidic protein and S100B, and the lack of the neuronal marker S100A. Notably, all the Ca2+ pathways present in astrocytes were detected in GL15 cells. In particular, oscillations in intracellular Ca2+ levels were recorded either spontaneously, or in the presence of ATP or glutamate (but not KCl). Immunolabelling assays and confocal microscopy, substantiated by Western blot analyses, revealed the presence of connexin43, a subunit of astrocyte gap-junction channels. The protein is organised in characteristic spots on the plasma membrane at cell-cell contact regions, and its presence and distribution depends on the differentiative status of the cell. Finally, a microinjection/dye-transfer assay, employed to determine gap-junction functionality, clearly demonstrated that the cells were functionally coupled, albeit to varying degrees, in differentiated and undifferentiated phenotypes.

Conclusions: In conclusion, results from this study support the use of the GL15 cell line as a suitable in vitro astrocyte model, which provides a valuable guide for studying glial physiological features at various differentiation phases.

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ATP-induced [Ca2+]i variations in differentiated GL15 cells. Panel A shows a single Ca2+ spike induced by 200 μM ATP (arrow) in 32% of differentiated cells (n = 69); addition of 2 μM thapsigargin (Tg) (arrow) evoked a further increase in [Ca2+]i. In 18% (12 out of 69) of differentiated cells, 200 μM ATP (arrow) induced Ca2+ oscillations, partially inhibiting the thapsigargin-induced Ca2+ increase (B). Time (s=seconds) is indicated on the abscissa; the ordinate gives the normalized fluorescence value (f/f0).
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Figure 5: ATP-induced [Ca2+]i variations in differentiated GL15 cells. Panel A shows a single Ca2+ spike induced by 200 μM ATP (arrow) in 32% of differentiated cells (n = 69); addition of 2 μM thapsigargin (Tg) (arrow) evoked a further increase in [Ca2+]i. In 18% (12 out of 69) of differentiated cells, 200 μM ATP (arrow) induced Ca2+ oscillations, partially inhibiting the thapsigargin-induced Ca2+ increase (B). Time (s=seconds) is indicated on the abscissa; the ordinate gives the normalized fluorescence value (f/f0).

Mentions: We also studied the membrane activating systems of GL15 cells by analysing, in single cells, the [Ca2+]i variations triggered by extracellularly applied stimuli for which the concentration and effect on astroglial cells are well known. Figures 3, 4, 5 and 6 show the temporal analysis of single cell [Ca2+]i variation expressed as normalized fluorescence values (see Materials and Methods).


Calcium-mediated transductive systems and functionally active gap junctions in astrocyte-like GL15 cells.

Mariggio MA, Mazzoleni G, Pietrangelo T, Guarnieri S, Morabito C, Steimberg N, Fano G - BMC Physiol. (2001)

ATP-induced [Ca2+]i variations in differentiated GL15 cells. Panel A shows a single Ca2+ spike induced by 200 μM ATP (arrow) in 32% of differentiated cells (n = 69); addition of 2 μM thapsigargin (Tg) (arrow) evoked a further increase in [Ca2+]i. In 18% (12 out of 69) of differentiated cells, 200 μM ATP (arrow) induced Ca2+ oscillations, partially inhibiting the thapsigargin-induced Ca2+ increase (B). Time (s=seconds) is indicated on the abscissa; the ordinate gives the normalized fluorescence value (f/f0).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC32183&req=5

Figure 5: ATP-induced [Ca2+]i variations in differentiated GL15 cells. Panel A shows a single Ca2+ spike induced by 200 μM ATP (arrow) in 32% of differentiated cells (n = 69); addition of 2 μM thapsigargin (Tg) (arrow) evoked a further increase in [Ca2+]i. In 18% (12 out of 69) of differentiated cells, 200 μM ATP (arrow) induced Ca2+ oscillations, partially inhibiting the thapsigargin-induced Ca2+ increase (B). Time (s=seconds) is indicated on the abscissa; the ordinate gives the normalized fluorescence value (f/f0).
Mentions: We also studied the membrane activating systems of GL15 cells by analysing, in single cells, the [Ca2+]i variations triggered by extracellularly applied stimuli for which the concentration and effect on astroglial cells are well known. Figures 3, 4, 5 and 6 show the temporal analysis of single cell [Ca2+]i variation expressed as normalized fluorescence values (see Materials and Methods).

Bottom Line: In particular, oscillations in intracellular Ca2+ levels were recorded either spontaneously, or in the presence of ATP or glutamate (but not KCl).The protein is organised in characteristic spots on the plasma membrane at cell-cell contact regions, and its presence and distribution depends on the differentiative status of the cell.In conclusion, results from this study support the use of the GL15 cell line as a suitable in vitro astrocyte model, which provides a valuable guide for studying glial physiological features at various differentiation phases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dipartimento di Scienze del Farmaco, Laboratorio di Fisiologia Cellulare, Università G, D'Annunzio, I-66013 Chieti. mariggio@unich.it

ABSTRACT

Background: It has been proposed that GL15, a human cell line derived from glioblastoma multiforme, is a possible astroglial-like cell model, based on the presence of cytoplasmic glial fibrillary acidic protein.

Results: The aim of this work was to delineate the functional characteristics of GL15 cells using various experimental approaches, including the study of morphology, mechanism of induction of intracellular Ca2+ increase by different physiological agonists, and the presence and permeability of the gap-junction system during cell differentiation. Immunostaining experiments showed the presence and localization of specific glial markers, such as glial fibrillary acidic protein and S100B, and the lack of the neuronal marker S100A. Notably, all the Ca2+ pathways present in astrocytes were detected in GL15 cells. In particular, oscillations in intracellular Ca2+ levels were recorded either spontaneously, or in the presence of ATP or glutamate (but not KCl). Immunolabelling assays and confocal microscopy, substantiated by Western blot analyses, revealed the presence of connexin43, a subunit of astrocyte gap-junction channels. The protein is organised in characteristic spots on the plasma membrane at cell-cell contact regions, and its presence and distribution depends on the differentiative status of the cell. Finally, a microinjection/dye-transfer assay, employed to determine gap-junction functionality, clearly demonstrated that the cells were functionally coupled, albeit to varying degrees, in differentiated and undifferentiated phenotypes.

Conclusions: In conclusion, results from this study support the use of the GL15 cell line as a suitable in vitro astrocyte model, which provides a valuable guide for studying glial physiological features at various differentiation phases.

Show MeSH
Related in: MedlinePlus