Administration of nicotinamide to rats produces specific dose-dependent inhibition of Na+-dependent phosphate transport across the renal brush-border membrane (BBM) and an increase in urinary excretion of phosphate. The intracellular mechanism of action of nicotinamide is not well established. As a step in this direction, the present studies determined whether nicotinamide was a rapid- or slow-acting regulator of the BBM phosphate transport system. Nicotinamide (0.5 g/kg) inhibited Na+-dependent BBM phosphate transport under conditions when de novo protein synthesis was inhibited by cycloheximide (1.0 mg/kg). Furthermore, the degree of inhibition was not different from that achieved by nicotinamide alone, suggesting that the action of nicotinamide does not require de novo protein synthesis. Studies on the time course of the onset of nicotinamide action revealed inhibition of BBM phosphate transport within 1 h after injection of nicotinamide, even in rats pretreated with cycloheximide. The rapid response to nicotinamide and its independence of de novo protein synthesis characterize nicotinamide as a rapid-acting regulator of the Na+-dependent phosphate transport system in renal BBM.
As with Methanococcus voltae [(1986) FEBS Lett. 200, 177-180], ATP synthesis in Methanobacterium thermoautotrophicum (AH) can be driven by the imposition of a sodium gradient, but only in the presence of a counterion. Monensin (but not SF6847) inhibits this synthesis. Methanogenic electron transfer-driven ATP synthesis, however, is insensitive to the combination of these two ionophores. In M. voltae, 117 I~M diethylstilbestrol effectively inhibits both membrane potential-and sodium gradient-driven ATP synthesis, but has no effect on ATP production coupled to methanogenesis. In Mb, thermoautotrophicurn (AII), a similar pattern of inhibition is exhibited by harmaline, an inhibitor of sodium-linked membrane transport systems. We conclude that ATP-driven sodium translocation and electron transfer-driven ATP synthesis are accomplished by separate entities, at least for these two only distantly related species ot methanogen.
MethanogenBioenergetics Na + pump A TPase
SUMMARY: A method is described for determining Na+-dependent solute uptake by an established cell line derived from opossum kidney. A description is included of the use of thyroid hormones and parathyroid hormone to study hormonal regulation of the Na+/phosphate cotransport system. The protocol uses cells grown in monolayer culture in 24-well plates, a system that facilitates rapid and efficient handling of many samples exposed to different conditions within the same experiment.
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