Abstract
PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Na(o) and 0.7 mM for K(o), is absolutely dependent on Cl(o) and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 ~ 650 nM) or calyculin A (EC50 ~ 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF an cotransport rate is biphasic, with an initial rapid ~2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 ~ 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (>2 days) leads to neurite formation and a maintained ~2- fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 10581-10589 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 269 |
| Issue number | 14 |
| State | Published - Apr 8 1994 |
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ASJC Scopus subject areas
- Biochemistry
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Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells. / Leung, Sara; O'Donnell, Martha E; Martinez, Anthony; Palfrey, H. Clive.
In: Journal of Biological Chemistry, Vol. 269, No. 14, 08.04.1994, p. 10581-10589.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Regulation by nerve growth factor and protein phosphorylation of Na/K/2Cl cotransport and cell volume in PC12 cells
AU - Leung, Sara
AU - O'Donnell, Martha E
AU - Martinez, Anthony
AU - Palfrey, H. Clive
PY - 1994/4/8
Y1 - 1994/4/8
N2 - PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Na(o) and 0.7 mM for K(o), is absolutely dependent on Cl(o) and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 ~ 650 nM) or calyculin A (EC50 ~ 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF an cotransport rate is biphasic, with an initial rapid ~2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 ~ 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (>2 days) leads to neurite formation and a maintained ~2- fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.
AB - PC12 cells possess a bumetanide-sensitive Na/K/2Cl cotransport system similar to that found in other cell types. Between 10-15% of the total 86Rb influx in these cells is mediated by this pathway under normal conditions. The cotransporter has affinities of 16.5 mM for Na(o) and 0.7 mM for K(o), is absolutely dependent on Cl(o) and is loop diuretic inhibitable (benzmetanide > bumetanide > piretanide > furosemide). The cotransporter can be activated (up to 8-fold) by cell shrinkage or (up to 4-fold) by treatment with the protein phosphatase inhibitors okadaic acid (EC50 ~ 650 nM) or calyculin A (EC50 ~ 8 nM). Cell shrinkage is followed by a bumetanide-sensitive regulatory volume increase as determined in cell sizing experiments. Calyculin A rapidly elevates normal cell volume in a diuretic-inhibitable manner. Cotransport activity and cell volume are also increased by nerve growth factor (NGF) treatment. The effect of NGF an cotransport rate is biphasic, with an initial rapid ~2.5-fold increase followed by a prolonged plateau, and is blocked by pretreatment of the cells with K252a (IC50 ~ 30 nM). By contrast, agents that raise cAMP or phorbol esters lead to an inhibition of cotransport, indicating that the NGF effect is not mediated by stimulation of either cAMP-dependent protein kinase or protein kinase C. Long term NGF treatment (>2 days) leads to neurite formation and a maintained ~2- fold increase in cotransport activity. Bumetanide treatment does not affect the ability of cells to extend neurites, nor is the growth rate of cells in normal medium affected by the diuretic. These results suggest that the cotransport system in PC12 cells is acutely regulated by protein phosphorylation and dephosphorylation as well as cell shrinkage and that cotransport activity may be up-regulated during neuronotypic differentiation.
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UR - http://www.scopus.com/inward/citedby.url?scp=0028365637&partnerID=8YFLogxK
M3 - Article
C2 - 8144646
AN - SCOPUS:0028365637
VL - 269
SP - 10581
EP - 10589
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 14
ER -