Limits...
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.

Show MeSH

Related in: MedlinePlus

Pattern of dye coupling in GL15 cell cultures Sub-confluent monolayers of undifferentiated GL15 cells (A and B) show the presence of dye-permeant junctional channels. When the monolayers reached confluence (C and D), the dye-spreading capacity of the cells was reduced. Almost no dye spreading is observed in differentiated confluent GL15 cultures (E and F). Fluorescence (B, D and F) and the corresponding phase contrast (A, C and E) photographs are taken on formaldehyde-fixed cells, 15 min after dye injection. The star symbol indicates the microinjected cell. Bar = 50 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC32183&req=5

Figure 7: Pattern of dye coupling in GL15 cell cultures Sub-confluent monolayers of undifferentiated GL15 cells (A and B) show the presence of dye-permeant junctional channels. When the monolayers reached confluence (C and D), the dye-spreading capacity of the cells was reduced. Almost no dye spreading is observed in differentiated confluent GL15 cultures (E and F). Fluorescence (B, D and F) and the corresponding phase contrast (A, C and E) photographs are taken on formaldehyde-fixed cells, 15 min after dye injection. The star symbol indicates the microinjected cell. Bar = 50 μm.

Mentions: i) Functional analysis of GL15 junctional coupling. A microinjection/dye-transfer assay was employed to determine GJIC strength in differentiated and undifferentiated GL15 monolayers, analysed at different culture densities. Cells from undifferentiated, proliferating (sub-confluent) monolayers were clearly shown to be junctionally coupled. When the saturation density (confluence) of the cultures was reached, a significant reduction of the cell coupling was observed (48% of the value found in the proliferating counterpart). In the case of differentiated, resting GL15 cells (confluent cultures), dye-transfer was almost completely restricted to the cells initially loaded with the dye (Table 2). The GJIC capacity of GL15 cultures is shown in Fig. 7.


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)

Pattern of dye coupling in GL15 cell cultures Sub-confluent monolayers of undifferentiated GL15 cells (A and B) show the presence of dye-permeant junctional channels. When the monolayers reached confluence (C and D), the dye-spreading capacity of the cells was reduced. Almost no dye spreading is observed in differentiated confluent GL15 cultures (E and F). Fluorescence (B, D and F) and the corresponding phase contrast (A, C and E) photographs are taken on formaldehyde-fixed cells, 15 min after dye injection. The star symbol indicates the microinjected cell. Bar = 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Pattern of dye coupling in GL15 cell cultures Sub-confluent monolayers of undifferentiated GL15 cells (A and B) show the presence of dye-permeant junctional channels. When the monolayers reached confluence (C and D), the dye-spreading capacity of the cells was reduced. Almost no dye spreading is observed in differentiated confluent GL15 cultures (E and F). Fluorescence (B, D and F) and the corresponding phase contrast (A, C and E) photographs are taken on formaldehyde-fixed cells, 15 min after dye injection. The star symbol indicates the microinjected cell. Bar = 50 μm.
Mentions: i) Functional analysis of GL15 junctional coupling. A microinjection/dye-transfer assay was employed to determine GJIC strength in differentiated and undifferentiated GL15 monolayers, analysed at different culture densities. Cells from undifferentiated, proliferating (sub-confluent) monolayers were clearly shown to be junctionally coupled. When the saturation density (confluence) of the cultures was reached, a significant reduction of the cell coupling was observed (48% of the value found in the proliferating counterpart). In the case of differentiated, resting GL15 cells (confluent cultures), dye-transfer was almost completely restricted to the cells initially loaded with the dye (Table 2). The GJIC capacity of GL15 cultures is shown in Fig. 7.

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