Activation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells by aluminum fluoride and phosphatase inhibitors.
The bumetanide-sensitive component of pHi recovery from an NH4Cl-induced acute alkaline load was used as a measure of Na(+)-K(+)-2Cl- cotransport activity in rat parotid acini. Acinar treatment with NaF/AlCl3 (15 mM NaF plus 10 microM AlCl3) induced a 5-fold stimulation in the initial rate of bumetanide-sensitive pHi recovery. This effect was dependent on NaF concentration (K1/2 approximately 7 mM) and was blunted in the presence of the Al3+ chelator desferal mesylate suggesting that it might be due to the aluminofluoride ion, AlF-4. NaF/AlCl3 treatment did not increase acinar intracellular cAMP levels but did result in an increase in intracellular calcium concentration (from 87 +/- 5 to 181 +/- 2 nM) and in acinar cell shrinkage (12 +/- 1%). But the stimulation of the Na(+)-K(+)-2Cl- cotransporter by NaF/AlCl3 persisted in acini which had been depleted of their intracellular Ca2+ stores. In these acini no effect of NaF/AlCl3 on intracellular calcium or cell volume was observed, indicating that stimulation of the cotransporter was not secondary to either of these phenomena. The effect of NaF/AlCl3 on the cotransporter was blocked by the protein kinase inhibitor K252a indicating the involvement of a protein phosphorylation event. This result is consistent with either NaF/AlCl3-dependent protein kinase activation or phosphatase inhibition. The stimulation of the cotransporter by NaF/AlCl3 was mimicked by the protein phosphatase inhibitor calyculin A; however, this effect was not blocked by K252a suggesting that a different protein kinase from that associated with NaF/AlCl3 may be involved. The data indicate that the Na(+)-K(+)-2Cl- cotransporter in this tissue is under tight regulatory control, in all likelihood via multiple protein kinase/phosphatase systems. The physiological roles of these regulatory events in modulating acinar fluid secretion driven by the Na(+)-K(+)-2Cl- cotransporter remain to be elucidated.
Paulais
M
M
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Turner
R J
RJ
D000535:Aluminum / Q000494:pharmacology*; D017607:Aluminum Compounds; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005459:Fluorides / Q000494:pharmacology*; D007473:Ion Channels / Q000378:metabolism*; D008297:Male; D010306:Parotid Gland / Q000187:drug effects / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D047428:Protein Kinase Inhibitors; D051381:Rats; D017208:Rats, Wistar; D012969:Sodium Fluoride / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters; D015854:Up-Regulation
D017607:Aluminum Compounds; D002352:Carrier Proteins; D007473:Ion Channels; D047428:Protein Kinase Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D012969:Sodium Fluoride; D000535:Aluminum; D010749:Phosphoprotein Phosphatases; D005459:Fluorides; C032311:aluminum fluoride