Document Detail

The regulation of Na/K/2Cl cotransport and bumetanide binding in avian erythrocytes by protein phosphorylation and dephosphorylation. Effects of kinase inhibitors and okadaic acid.
MedLine Citation:
PMID:  2147426     Owner:  NLM     Status:  MEDLINE    
The Na/K/2Cl cotransport system in the avian erythrocyte can be activated by agents that raise intracellular cAMP suggesting the involvement of cAMP-dependent protein kinase (cAMP-PK) in its regulation. Another group of stimuli including fluoride and hypertonicity stimulate cotransport via cAMP-independent means. To further investigate the role of phosphorylation in these processes, we examined the effects of protein kinase inhibitors of 8 (p-Cl-phenylthio)-cAMP (cpt-cAMP), fluoride and hypertonic activation of cotransport in duck red cells, and [3H]bumetanide binding to isolated membranes. Preincubation of cells with the kinase inhibitors K-252a (Ki approximately 1.6 microM) and H-9 (Ki approximately 100 microM) blocked cpt-cAMP activation of bumetanide-sensitive 86Rb influx and bumetanide binding. These inhibitors also led to a rapid deactivation of cotransport and decrease in bumetanide binding when added to cells maximally stimulated by cpt-cAMP. K-252a and H-9 inhibited cotransport activation by cAMP-independent stimuli, but 10-fold higher concentrations were required, implying the involvement of a cAMP-independent phosphorylation process in the mechanism of action of these agents. Removal of stimuli that elevate cAMP leads to a rapid reversal of cotransport indicating the presence of active protein phosphatases in these cells. The protein phosphatase inhibitor okadaic acid (OA, EC50: 630 nM) stimulated both Na/K/2Cl cotransport and bumetanide binding to membranes. As with fluoride and hypertonic stimulation, the OA effect was inhibited only at relatively high concentrations of K-252a. Phosphorylation of the membrane skeletal protein goblin (Mr 230,000) at specific cAMP-dependent sites was used as an in situ marker for the state of activation of cAMP-PK. Goblin phosphorylation at these sites was increased by norepinephrine and cpt-cAMP and rapidly reversed by K-252a and H-9, confirming that both inhibitors do block cAMP-PK activity. While OA markedly increased overall phosphorylation of many erythrocyte membrane proteins, including goblin, it did not affect goblin phosphorylation at specific cAMP-dependent sites. These results implicate a cAMP-independent protein kinase in the mediation of the OA effect on cotransport and bumetanide binding. The bumetanide-binding component of the avian erythrocyte cotransporter, an Mr approximately 150,000 protein that can be photolabeled with the bumetanide analog [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)-benzoic acid was found to be a phosphoprotein. These results strongly support the hypothesis that phosphorylation and dephosphorylation, possibly of the Na/K/2Cl cotransporter itself, regulates the activity of
E B Pewitt; R S Hegde; M Haas; H C Palfrey
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  265     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1990 Dec 
Date Detail:
Created Date:  1991-01-17     Completed Date:  1991-01-17     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  20747-56     Citation Subset:  IM    
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
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MeSH Terms
Blood Proteins / metabolism
Bumetanide / blood*
Carbazoles / pharmacology
Carrier Proteins / blood*
Chlorides / blood*
Erythrocyte Membrane / drug effects,  metabolism
Erythrocytes / drug effects,  metabolism*
Ethers, Cyclic / pharmacology*
Indole Alkaloids
Isoquinolines / pharmacology
Membrane Proteins / metabolism
Okadaic Acid
Potassium / blood*
Protein Kinase Inhibitors
Protein Kinases / blood*
Sodium / blood*
Sodium-Potassium-Chloride Symporters
Grant Support
Reg. No./Substance:
0/Ankyrins; 0/Blood Proteins; 0/Carbazoles; 0/Carrier Proteins; 0/Chlorides; 0/Ethers, Cyclic; 0/Indole Alkaloids; 0/Isoquinolines; 0/Membrane Proteins; 0/Protein Kinase Inhibitors; 0/Sodium-Potassium-Chloride Symporters; 0/Sulfonamides; 28395-03-1/Bumetanide; 7440-09-7/Potassium; 7440-23-5/Sodium; 78111-17-8/Okadaic Acid; 84468-17-7/N-(2-aminoethyl)-5-isoquinolinesulfonamide; 97161-97-2/K 252; EC 2.7.-/Protein Kinases

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