Abstract
The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.
Highlights
ATPase transportactivity in freshly isolated rat hepa- Freshly prepared suspensions of isolated rat hepatocytes tocytes were investigated by measuring the ouabain- have beenshown to retain metabolic activity [12, 13], to sensitive, active uptake of"Rb'
Preparation of Isolated Hepatocytes andIncubation Proce-These results indicate that monovalent cation trans- dures-Hepatocytes were isolated from fed male Wistar rats (120-150 port is influenced by insulin and glucagon in isolated g) by collagenase dissociation of the liver as previously described (13, rat hepatocytesI.n contrastto glucagon, which appears18)
Effect of Insulin and Glucagononthe Time Course of 86Rb' Uptake (Fig. 1)-Total 86Rb+uptake by isolated hepatocytes was linear for about 2 min and reached a maximal value after 60 to 120 min
Summary
Effect of Insulin and Glucagononthe Time Course of 86Rb' Uptake (Fig. 1)-Total 86Rb+uptake by isolated hepatocytes was linear for about 2 min and reached a maximal value after 60 to 120 min. Each point is the mean of Stimulation of 86Rb+Uptake-The stimulatory effect of intriplicate determinations within one typical experiment. Comparison between Hormone Binding and Stimulation of "Rb+ Uptake-At 37 "C in isolated hepatocytes, insulin binds predominantly to one class of high affinity receptors with an apparent Kd of0.6 nM [16].When the percentage of maximal insulin effect on "Rb+ uptake was plotted against the percentage of maximal insulin binding to total receptor sites at 37 "C the relationship between binding and effect deviated from linearity only slightly, with 50% of maximal effect occurring at about 30% oftotal receptor occupancy (Fig. 5, left). When the same analysis was made only taking into account insulin binding tothe high affinity site, a linear
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