The effects of metabolism on Na(+)‐K(+)‐Cl‐ co‐transport in ferret red cells.

Abstract

1. The effects of altering metabolism on Na(+)-K(+)-Cl- co-transport were studied in ferret red cells. Na(+)-K(+)-Cl- co-transport was measured as the bumetanide-sensitive uptake of 86Rb. 2. Glucose, but not inosine or adenosine, sustained metabolism and maintained cell ATP content ([ATP]i) at the physiological level. [ATP]i could be reduced by prolonged incubation of cells in a substrate-free medium or more quickly by incubating cells with 2-deoxyglucose or with a mixture of iodoacetamide and glucose. 3. Na(+)-K(+)-Cl- co-transport activity was inhibited when [ATP]i was reduced to below 100 mumol (1 cell)-1 by starvation or by treatment with 2-deoxyglucose. However, a unique relationship between [ATP]i and activity could not be found. [ATP]i and the method and time course of ATP depletion all influenced activity. The inhibition of Na(+)-K(+)-Cl- co-transport, caused by reducing [ATP]i could be partially reversed by restoring [ATP]i to normal. 4. Increasing the concentration of intracellular ionized magnesium [( Mg2+]i) did not stimulate co-transport activity in ATP-depleted cells. This contrasts with the substantial stimulation seen in cells with normal [ATP]i. 5. Vanadate stimulated Na(+)-K(+)-Cl- co-transport activity in ATP-depleted cells but not in cells with normal [ATP]i. Fluoride did not affect activity at any [ATP]i. 6. The effects of some sulphydryl reagents on Na(+)-K(+)-Cl- co-transport were also examined. n-Ethylmaleimide (1 mM) inhibited Na(+)-K(+)-Cl- co-transport while it stimulated bumetanide-resistant potassium transport. Dithiothreitol (1 mM) did not affect activity. Iodoacetamide (6 mM) appeared to reduce the inhibition of cotransport activity seen at low [ATP]i but also greatly increased cell fragility. 7. The data suggest that activity of the Na(+)-K(+)-Cl- co-transport system is controlled by a cycle of phosphorylation and dephosphorylation with the phosphorylated form being active. Phosphorylation and transport appear to be almost maximal in ferret red cells with normal [ATP]i. Reduction of [ATP]i may allow changes in phosphatase activity to manifest as changes in transport rate. Differences in the balance between phosphorylation and dephosphorylation may explain tissue-dependent variations in the response of the system to various stimuli.