SU-MMARY1. Uptake and release of radiophosphate were measured in desheathed rabbit vagus nerve.2. During incubation in Locke containing 0-2 mM-[32P]phosphate a slow labelling of water soluble compounds of the nerve was found; the labelling of the non water soluble compounds was much smaller. During washing with inactive Locke, the label was almost entirely released from the water soluble compounds; the radioactivity of these compounds was therefore used as the basis for the calculation of the efflux rate constants.3. The efflux of radiophosphate increased with increasing phosphate concentration of the washing fluid.4. A similar effect of external phosphate on the efflux of radiophosphate was seen when the phosphate concentration was suddenly changed. The rate constants were in 0 02 mM-phosphate PM29 x 10-m d-1, in 0-2 mM 1-95 x 10-3 min-I, and in 2 mm 3-21 x 10-3 min-' at 37 'C. After changing the external solution the efflux reached a new level with a time constant of about 9 mi.5. Addition of arsenate also increased the efflux of radiophosphate; on a molar basis the effect of arsenate was slightly smaller than the effect of phosphate. the ratio between the Na dependent effluxe8 in 2 and 0-2 mM-phosphate was approximately equal to the ratio between the Na dependent influxes in 2 and 0*2 mMphosphate.10. The efflux of 22Na had a rate constant of 0 050 min-' in Locke and 0x046 minin Tris-Locke.11. It is concluded that part of the efflux of phosphate is mediated by a 508 J. FERRERO AND OTHERS Na-dependent transport system; the system appears to be able to exchange phosphate between the inside and the outside and to mediate net movements of phosphate in both directions.

Phosphate efflux was measured as the fractional rate of loss of radioactivity from rabbit vagus loaded with radiophosphate. The effects of changes in extracellular calcium and of lanthanum have been investigated. In Locke solution with normal, 0.9 mM, calcium and without phosphate, the fractional rate of loss was 1.62 X 10(-3) min-1 at 120 min after the beginning of the washing period and fell slowly (9% hr-1) during washing from 2 to 6 hr. Addition of calcium to the Locke solution produced a transient increase followed by a reversible maintained increase in phosphate efflux. The latter was 40 and 75% above efflux in normal calcium for 20 and 50 mM calcium, respectively. Removal of calcium, with or without addition of EGTA, produced only a transient increase in phosphate efflux, with no subsequent maintained change. Addition of low concentrations of lanthanum produced a reversible inhibition of phosphate efflux. Half-maximal inhibition was at 3.5 micro M lanthanum and appeared to be due to binding of lanthanum to more than one, probably two, sites. Measurements of inhibition by lanthanum at different calcium concentrations did not indicate any competition between calcium and lanthanum. It is suggested that a least a part of phosphate efflux depends on internal calcium and that lanthanum acts by preventing release of phosphate from the phosphate transport mechanism.

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Summary. The efflux of phosphate was measured in rabbit vagus nerve loaded with radiophosphate. The efflux was found to depend on the K concentration of the bathing solutions; increasing the K from 5.6 up to 150 mM produced a maximal lowering of 28% ; K-free solution produced a transient increase whose peak was 86% above the normal efflux. In the presence of Na, the K-free effect could be repeated; in Na-free solution, it was found only for the first application of the K-free solution. The phosphate efflux was not altered when K was replaced by Rb; replacement with Cs showed that this ion only partially mimics the effect of K.The results suggest that the transient increase in phosphate effiux is due to release of label from a K-dependent saturable binding site, which is distinct from the main intracellular pool. The binding site appears to be labeled from the inside by the Nadependent phosphate efflux previously described. It may correspond to the phosphorylation of membrane phospholipids. A mathematical model of this system is developed and curves simulated by an analog computer are compared to the experimental results.Measurements of the membrane potential and the internal inorganic phosphate showed that the effect of K on the phosphate efflux could not be explained by changes in the membrane potential or in the internal phosphate pool.We have shown previously that a large proportion of the transmembrane fluxes of inorganic phosphate in nonmyelinated nerve fibers is mediated by a saturable Na-dependent mechanism (Armor et al., 1976;Ferrero et al., 1978). This mechanism is different from the phosphate transport in other tissues, e.g., erythrocytes (Rothstein, Cabantchick & Knauf, 1976), mitochondria (Banerjee et al., 1977) or Escherichia coli (Rosenberg, Gerdes & Harold, 1979).In the course of further investigations on the phosphate efflux, a transient release of phosphate was observed when the potassium of the incubation solution was withdrawn. Conversely, when the potassium was increased, the efflux of phosphate was diminished. In the present study, we have analyzed these phenomena in more detail. Our results suggest that the phosphate released in the absence of K originates from a specific K-dependent phosphate binding site. Materials and MethodsDesheathed rabbit vagus nerves were mounted in a polyethylene tube which was perfused with 32p-phosphate Locke for 150 rain at 37 ~ The preparation then was washed with inactive Locke solution, and the effluent was collected and counted. At the end of the experiment the preparation was homogenized in 0.1 g triethanolamine buffer (pH 8). The homogenate was mixed with chloroform, centrifuged at 3,000 xg for 15 min and the activity of the water-soluble fraction was then counted. These counts were used for the calculation of the efflux rate constant. The composition of the Locke was (raM): 154 NaC1, 5.6 KC1, 0.9 CaC12, 0.5 MgCI2, 5 glucose, 0.2 Na2HPO4-NaH2PO4, 1 Tris. Potassium isethionate (Eastman-Kodak) was added to prepare K-rich Locke. In Na-free solutions, sodium wa...

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