Swelling-induced activation of Na+,K+,2Cl- cotransport in C6 glioma cells: kinetic properties and intracellular signalling mechanisms.
Swelling of C6 glioma cells in hypotonic medium (180 mOsm) results in two- to three-fold activation of K+ (86Rb+) influx suppressed by 10 microM bumetanide. Bumetanide-sensitive transport of 86Rb+ is dependent on extracellular K+, Na+ and Cl- both in iso-osmotic conditions and under hypo-osmotic shock, supporting the notion that it is mediated by Na+,K+,2Cl- cotransport. Inhibitors of protein kinase C (10 microM polymyxin B and l microM staurosporine) had no significant effect on basal cotransport but reduced its hypotonic stimulation by 70-80%. Similar results were obtained with calmodulin antagonist R24571 (10 microM), indicating Ca2+/calmodulin-dependence of the process. Influence of polymyxin B and R24571 was not additive. Swelling-activated Na+,K+,2Cl- cotransport was also suppressed by protein kinase C activator PMA (l microM). By contrast, preincubation of cells with inhibitors of protein phosphatases (100 microM vanadate, 5 mM fluoride and 0.5 microM okadaic acid) activated greatly the bumetanide-sensitive 86Rb+ uptake in isotonic conditions, while a subsequent hypotonic swelling led to smaller or no increment. These results indicate the involvement of Ca2+/calmodulin-dependent staurosporine/polymyxin B-sensitive protein kinase other than protein kinase C in swelling-induced activation of Na+,K+,2Cl- cotransport in glial cells.
Mongin
A A
AA
Institute of Photobiology, Belarussian Academy of Sciences, Minsk, Belarus, mongin@bas07.basnet.minsk.by
Aksentsev
S L
SL
Orlov
S N
SN
Kvacheva
Z B
ZB
Mezen
N I
NI
Fedulov
A S
AS
Konev
S V
SV
D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D048429:Cell Size; D002712:Chlorides / Q000378:metabolism / Q000494:pharmacology; D004232:Diuretics / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism; D007038:Hypotonic Solutions / Q000494:pharmacology; D007474:Ion Exchange; D007700:Kinetics; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D002712:Chlorides; D004232:Diuretics; D007038:Hypotonic Solutions; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D010042:Ouabain; D012964:Sodium; D012413:Rubidium; D011188:Potassium