Irreversible inhibition of renal Na(+)-Pi cotransporter by alpha-bromophosphonoacetic acid.
We investigated the suitability of alpha-bromophosphonoacetic acid (alpha-BrPAA) to act as a possible irreversible inhibitor of Na(+)-dependent transport of Pi across renal brush-border membrane (BBM). When added directly into the Pi uptake medium, alpha-BrPAA causes specific, competitive [apparent inhibition constant (Ki) = 0.33 mM; no change in maximum velocity (Vmax)], and reversible (by washing) inhibition of Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by BBM vesicles (BBMV). Next, BBMV were preincubated with 5 mM alpha-BrPAA in alkaline (pH 9) medium for 30 min, then twice washed by 1:100 dilution and recentrifugation, and tested for transport and other properties. This preincubation of BBMV with alpha-BrPAA in alkaline medium resulted in a different type of inhibition [lower Vmax; no change in Michaelis constant (Km)] of the Na+ gradient-dependent uptake of 32Pi, whereas the uptakes of D-[3H]glucose and other solutes were not altered. This inhibition of Pi transport was not reversed by dilution and washing of BBMV. The BBMV Na(+)-dependent binding of [14C]phosphonoformic acid, but not of [3H]phlorizin, was decreased; activities of BBM marker enzymes were not changed. Results suggest that alpha-BrPAA binds onto the same locus on luminal surface of BBM on which Pi and Na+ bind and inhibits Na(+)-Pi cotransporter similar to phosphonoformic acid. Furthermore, after a 30-min incubation in alkaline medium, alpha-BrPAA apparently forms a more stable association with BBM in the vicinity of the Na(+)-Pi cotransporter. We thus suggest that alpha-BrPAA acts under these conditions as an apparently irreversible inhibitor of Na(+)-Pi cotransporter in BBM.(ABSTRACT TRUNCATED AT 250 WORDS)
Szczepanska-Konkel
M
M
Department of Physiology and Biophysics, Mayo Clinic and Foundation, Mayo Medical School, Rochester, Minnesota 55905.
Hoppe
A
A
Lin
J T
JT
Dousa
T P
TP
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002951:Citrates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007672:Kidney Cortex / Q000502:physiology*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D010710:Phosphates / Q000378:metabolism; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D011392:Proline / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000494:pharmacology; D050608:Sodium-Phosphate Cotransporter Proteins; D013386:Succinates / Q000378:metabolism; D027981:Symporters
D002352:Carrier Proteins; D002951:Citrates; D010710:Phosphates; D050608:Sodium-Phosphate Cotransporter Proteins; D013386:Succinates; D027981:Symporters; C063896:2-bromophosphonoacetic acid; D011392:Proline; D012964:Sodium; D005947:Glucose; D010746:Phosphonoacetic Acid