Stimulation of Protein Kinase C Rapidly Reduces Intracellular Na+ Concentration via Activation of the Na+Pump in OK Cells
- 1College of Pharmacy, University of Houston, Houston, Texas 77204-5515 (C.H.P., M.F.L.), 2Department of Physiology, Texas Tech University, Lubbock, Texas 79430 (T.A.P.), and 3Department of Anatomy and Cell Biology, State University of New York at Brooklyn, New York 11203 (A.R.C.)
Abstract
Na+ reabsorption is regulated in proximal tubules by hormones that stimulate protein kinase C (PKC). To determine whether stimulation of PKC causes a reduction in intracellular Na+concentration ([Na+]i) that might link Na+ pump activation to increased Na+reabsorption, [Na+]i was measured in kidney cells loaded with the Na+-sensitive fluorescent indicator SBFI. Rapid digital imaging fluorescence microscopy determinations were performed in epithelial kidney cells transfected with the rodent Na+ pump α1 cDNA. In 42 determinations, the basal [Na+]i was 19.7 ± 2.4 mm. Stimulation of PKC reduced the [Na+]i to 5.6 ± 0.6 mm in ∼10 sec. This drastic change in [Na+]i requires a transient 74–120-fold increase in Na+ pump activity. After the new steady state [Na+]i is reached, the Na+ pump is 58% activated. The entry of Na+ into the cells is not affected by stimulation of PKC; therefore, the reduction in [Na+]i is exclusively dependent on activation of the Na+ pump. Accordingly, PKC stimulation does not affect the [Na+]i of cells expressing a mutant Na+ pump that is not stimulated by PKC. The decrease in [Na+]i observed in cells transfected with the rodent Na+ pump α1 cDNA is large and sufficiently fast that it is expected to stimulate rapidly passive Na+-influx into the cells, thereby accounting for the observed PKC-induced stimulation of Na+ reabsorption.
Footnotes
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Send reprint requests to: Dr. Carlos H. Pedemonte, University of Houston, Calhoun 4800, Houston, TX 77204–5515. E-mail:pedemonte{at}jetson.uh.edu
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↵1 In suspension, OK cells have a diameter of ∼17 μm. Using this value and the equation to calculate the volume of an sphere, a volume of 2.4 pl was calculated. It was assumed that attached cells have the same intracellular volume as cells in suspension.
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↵2 This value was calculated from determinations of protein of a known number of cells.
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This work was supported by grants from the National Science Foundation (C.H.P.) and the National Institutes of Health [DK52273 (C.H.P.), RR19799 (T.A.P.), and DC01804 (A.R.C.)] and a limited grant-in-aid from the University of Houston (C.H.P.).
- Abbreviations:
- PKC
- protein kinase C
- DMSO
- dimethylsulfoxide
- [Na+]i
- intracellular sodium concentration
- PMA
- phorbol-12-myristate 13-acetate
- PDD
- phorbol-12,13-didecanoate
- MIA
- 5-(N-methyl-N-isobutyl)-amiloride
- DMEM-10
- Dulbecco’s modified Eagle’s medium with 10% calf serum and antibiotics
- PVDF
- polyvinylidene difluoride
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- BSA
- bovine serum albumin
- SDS
- sodium dodecyl sulfate
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- Received February 14, 1997.
- Accepted March 27, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
