Effects of okadaic acid and intracellular Cl- on Na(+)-K(+)-Cl- cotransport.
The Na(+)-K(+)-Cl- cotransporter of the squid giant axon requires ATP and is inhibited by intracellular Cl- (Cli-) in a concentration-dependent manner ([Cl-]i > or = 200 mM completely inhibits the cotransporter). In the present study we address the question of whether inhibition of cotransport by Cli- is due to a Cl(i-)-induced increase of protein phosphatase activity. Intracellular dialysis was used to apply the phosphatase inhibitor okadaic acid (OKA) under conditions of [Cl-]i at 0, 150, or 300 mM during measurement of cotransporter-mediated unidirectional Cl- influx into axons. At 0 mM [Cl-]i, the application of 250 nM OKA had no effect on the cotransport-mediated Cl- influx when axons were dialyzed with the normal intracellular ATP concentration ([ATP]i = 4 mM). Reduction of [ATP] to 50 microM resulted in a significant decrease of the bumetanide-sensitive CL- influx, which could be partially reversed by OKA treatment. Similarly, in ATP-limited axons with [Cl-]i at 150 mM, cotransporter influx was partially stimulated by treatment with OKA. However, axons dialyzed with 300 mM [Cl-]i ([ATP]i = 50 microM) had no measurable cotransport influx, nor was subsequent treatment with OKA able to induce a cotransport-mediated Cl- influx. We conclude that the inhibition of cotransport caused by Cli- is not the result of an increase in the OKA-sensitive protein phosphatase activity.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001369:Axons / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D049832:Decapodiformes; D004988:Ethers, Cyclic / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D019319:Okadaic Acid; D009994:Osmolar Concentration; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D002712:Chlorides; D004988:Ethers, Cyclic; D028021:Sodium-Potassium-Chloride Symporters; D019319:Okadaic Acid; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases