Abstract
We investigated interactions of phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and other phosphonyl derivatives with the Na+ gradient [Na+ extravesicular greater than Na+ intravesicular; Nao+ greater than Na+i]-dependent transport system for phosphate (Pi) in renal cortical brush border membrane vesicles (BBMV). PFA and PAA inhibited in a dose-dependent manner the Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by rat kidney BBMV. PFA was a more potent inhibitor than PAA while phosphonopropionic acid, hydroxymethylphosphonic acid, and phenylphosphonic acid had no effect on Pi transport. The inhibitory effect of PFA was competitive (Ki approximately equal to 4.6 X 10(-4) M) and reversible upon dilution. The uptake of Pi by BBMV in the absence of Na+ gradient [Nao+ = Na+i] was also inhibited by PFA. The PFA had no effect on uptake of L-[3H]proline, D-[3H]glucose, or 22Na+ by BBMV nor did it alter intravesicular volume of BBMV. The relative (%) extent of inhibition by PFA was not altered by changes in the extravesicular pH or changes in the steepness of the Na+ gradient [Nao+ greater than Na+i]. The inhibition of PFA was analogous in renal BBMV from rats, mice, rabbits, or dogs. Unlike other known inhibitors of brush border membrane (BBM) transport of Pi, e.g. arsenate, NAD, and ethane-1-hydroxy-1,1-diphosphonate, PFA and PAA had no inhibitory effect on BBM-bound or solubilized alkaline phosphatase. Also, PFA did not interfere with the activity of renal cortical (Na-K)ATPase. Administration of PFA (0.5 g/kg/day, intraperitoneally) to thyroparathyroidectomized rats fed a low Pi diet elicited an increase in urinary excretion of Pi, but did not change the excretion of Na+, K, and Ca2+. The results show that the PFA, and to a lesser degree PAA, are specific competitive inhibitors of the Na+-Pi cotransport in renal cortical BBM and are suitable probes for studies of this transport system.
Highlights
From the Nephrology Research Unit,Division of Nephrology and Internal Medicine, Department of Physiology, Mayo Clinic, Rochester, Minnesota 55905
The uptake of Piby BBMVin the absence position of Na+-Pi cotransporter in renal brush border membrane (BBM) wouldbe considerably aided by availability of specific inhibitorsfs), of Na+ gradient [Na", = Na+J was inhibited by suitable for thestudies of this transport system both in uitro
This article must be hereby marked "adverinhibitor of the renal Na+ gradient-dependent transport of Pi across.BBM, we considered and studied the properties of alkylphosphonic compounds,namelyphosphonocarboxylic acids [14]
Summary
Wereprepared from renal cortical tissue of adult male Sprague-Dawley rats of average body weight of about 220 g. This mg of protein/tube) was suspended in a medium containing 300 mM plasma membrane preparationcontains mainly basolateral mem- sucrose buffered with Tris-HEPES (pH 7.5), in a total volume of 150 branes, as indicated by marked enrichment (9-fold)of (Na-K)ATPase pl, and incubated with tested compounds at a final concentration of and a decrease in alkaline phosphatase activity, as well as bylow 1or 5 mM. Full size photocopies are included in the microfilm on 32Pitransport were explored by measuring the 32Piuptake by BBMV at theinitial (5 s)period, and resultswere evaluated by the Lineweaver-Burk double-reciprocalplot (Fig. 4) Both PFA and PAA increased apparent K, for the 32Piuptake without influencing the apparent Vma. The results of kinetic edition of the Journal thaits available from Waverly Press.
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