Na+/K+/Cl- cotransport is stimulated by a Ca(++)-calmodulin-mediated pathway in BALB/c 3T3 fibroblasts.
In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl- cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl- cotransport was stimulated by the growth factors EGF, TGF-alpha, IGF-1, and IGF-2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++. In addition, direct activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) did not affect the Na+/K+/Cl- cotransport activity of quiescent cells. The Na+/K+/Cl- cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl- cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl- cotransport is stimulated predominantly by a protein kinase C-independent mechanism in BALB/c 3T3 fibroblasts. Both the intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine (CP), blocked serum- and mitogen-stimulated Na+/K+/Cl- cotransport. These results suggest that the Na+/K+/Cl- cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca(++)-calmodulin-mediated pathway in the synchronized BALB/c 3T3 fibroblasts.
Snyder
D
D
Department of Medical Biophysics and Nuclear Medicine, Hadassah University Hospital, Jerusalem, Israel.
Atlan
H
H
Markus
M
M
Panet
R
R
D016475:3T3 Cells; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D002746:Chlorpromazine / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D011188:Potassium / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology*
D002121:Calcium Channel Blockers; D002147:Calmodulin; D002352:Carrier Proteins; D002712:Chlorides; D006133:Growth Substances; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D014268:Trifluoperazine; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D004815:Epidermal Growth Factor; D005707:Gallic Acid; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D012964:Sodium; D011493:Protein Kinase C; D012413:Rubidium; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium; D002118:Calcium; D002746:Chlorpromazine