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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
34
|
| pubmed:dateCreated |
1991-1-17
|
| pubmed:abstractText |
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
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| pubmed:grant | |
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Ankyrins,
http://linkedlifedata.com/resource/pubmed/chemical/Blood Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Bumetanide,
http://linkedlifedata.com/resource/pubmed/chemical/Carbazoles,
http://linkedlifedata.com/resource/pubmed/chemical/Carrier Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Chlorides,
http://linkedlifedata.com/resource/pubmed/chemical/Ethers, Cyclic,
http://linkedlifedata.com/resource/pubmed/chemical/Indole Alkaloids,
http://linkedlifedata.com/resource/pubmed/chemical/Isoquinolines,
http://linkedlifedata.com/resource/pubmed/chemical/Membrane Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/N-(2-aminoethyl)-5-isoquinolinesulfo...,
http://linkedlifedata.com/resource/pubmed/chemical/Okadaic Acid,
http://linkedlifedata.com/resource/pubmed/chemical/Potassium,
http://linkedlifedata.com/resource/pubmed/chemical/Protein Kinase Inhibitors,
http://linkedlifedata.com/resource/pubmed/chemical/Protein Kinases,
http://linkedlifedata.com/resource/pubmed/chemical/Sodium,
http://linkedlifedata.com/resource/pubmed/chemical/Sodium-Potassium-Chloride Symporters,
http://linkedlifedata.com/resource/pubmed/chemical/Sulfonamides,
http://linkedlifedata.com/resource/pubmed/chemical/staurosporine aglycone
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Dec
|
| pubmed:issn |
0021-9258
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
5
|
| pubmed:volume |
265
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
20747-56
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| pubmed:dateRevised |
2011-11-17
|
| pubmed:meshHeading |
pubmed-meshheading:2147426-Animals,
pubmed-meshheading:2147426-Ankyrins,
pubmed-meshheading:2147426-Birds,
pubmed-meshheading:2147426-Blood Proteins,
pubmed-meshheading:2147426-Bumetanide,
pubmed-meshheading:2147426-Carbazoles,
pubmed-meshheading:2147426-Carrier Proteins,
pubmed-meshheading:2147426-Chlorides,
pubmed-meshheading:2147426-Erythrocyte Membrane,
pubmed-meshheading:2147426-Erythrocytes,
pubmed-meshheading:2147426-Ethers, Cyclic,
pubmed-meshheading:2147426-Indole Alkaloids,
pubmed-meshheading:2147426-Isoquinolines,
pubmed-meshheading:2147426-Kinetics,
pubmed-meshheading:2147426-Membrane Proteins,
pubmed-meshheading:2147426-Okadaic Acid,
pubmed-meshheading:2147426-Phosphorylation,
pubmed-meshheading:2147426-Potassium,
pubmed-meshheading:2147426-Protein Kinase Inhibitors,
pubmed-meshheading:2147426-Protein Kinases,
pubmed-meshheading:2147426-Sodium,
pubmed-meshheading:2147426-Sodium-Potassium-Chloride Symporters,
pubmed-meshheading:2147426-Sulfonamides
|
| pubmed:year |
1990
|
| pubmed:articleTitle |
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.
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| pubmed:affiliation |
Department of Pharmacological and Physiological Sciences, University of Chicago, Illinois 60637.
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| pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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