Quantitation of the Na(+)-Pi cotransporter in renal cortical brush border membranes. [14C]phosphonoformic acid as a useful probe to determine the density and its change in response to parathyroid hormone.
To determine the density of Na(+)-Pi symporters in brush border membranes (BBM) from rat renal cortex, [14C] phosphonoformic acid [( 14C] PFA), a competitive inhibitor of Na(+)-Pi cotransport, was employed as a probe. The [14C]PFA binding was measured in BBM vesicles (BBMV) under equilibrated conditions (extra-vesicular Na+, K+, and H+ = intravesicular Na+, K+, and H+) to avoid modulatory effects of these solutes. BBMV were preincubated in media without or with addition of molar excess of Pi (greater than 20 times) to determine the Pi-protectable PFA-binding sites, and then [14C] PFA binding was determined. Only the [14C]PFA binding in the presence of Na+ displaceable by an excess of Pi was saturated and was independent of intravesicular volume of BBMV. This value denoted as "Pi-protectable Na(+)-[14C]PFA binding," was analyzed by Scatchard plot showing BmaxPFA = 375 +/- 129 pmol of PFA/mg protein, KDPFA = 158 +/- 18 microM; the Hill coefficient was congruent to 1. For Na(+)-dependent binding of [3H]phlorizin, in the same BBMV, Bmax = 310 +/- 37 pmol/mg protein and KD V 2.2 +/- 0.5 microM. BBMV prepared from cortex of thyroparathyroidectomized rats infused with phosphaturic doses of parathyroid hormone (PTH) were compared with vehicle-infused controls. Administration of PTH resulted in decrease of BmaxPFA (-38%) and of Na(+)-gradient-dependent uptake of 32Pi (-35%), but KDPFA was not changed. Neither BmaxPhl and KDPhl for Na(+)-phlorizin binding, nor the Na(+)-gradient-dependent uptake of [3H]D-glucose differed between PTH-treated and control rats. We conclude: (a) measurement of Pi-protectable Na(+)-[14C]PFA binding determines numbers and affinity of Na(+)-Pi symporters in renal BBMV; (b) the affinity of PFA for Na(+)-Pi symporter is similar to apparent affinity for Pi (KmPi), as determined from measurements of Na(+)-gradient-dependent 32Pi uptake by BBMV; (c) both Na(+)-Pi symporter and [Na+]D-glucose symporters are present within renal BBM in a similar range of density; (d) PTH decreases the number of Na(+)-Pi cotransporters in BBMV commensurate with the parallel decrease of Na(+)-gradient-dependent Pi transport, whereas the affinity of Na(+)-Pi symporters for Pi is not changed. These observations support the hypothesis that PTH decreases capacity for Na(+)-dependent Pi reabsorption by internalization of Na(+)-Pi symporters in BBM of renal proximal tubules.
Hoppe
A
A
Nephrology Research Unit, Mayo Clinic, Mayo Medical School, Rochester, Minnesota 55905.
Lin
J T
JT
Onsgard
M
M
Knox
F G
FG
Dousa
T P
TP
D000818:Animals; D001794:Blood Pressure; D002352:Carrier Proteins / Q000378:metabolism*; D017245:Foscarnet; D005919:Glomerular Filtration Rate; D007672:Kidney Cortex / Q000737:chemistry* / Q000502:physiology; D008297:Male; D008871:Microvilli / Q000737:chemistry* / Q000502:physiology; D009943:Organophosphorus Compounds / Q000097:blood / Q000652:urine; D009994:Osmolar Concentration; D010281:Parathyroid Hormone / Q000502:physiology*; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D051381:Rats; D011919:Rats, Inbred Strains; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D013696:Temperature
D002352:Carrier Proteins; D009943:Organophosphorus Compounds; D010281:Parathyroid Hormone; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D017245:Foscarnet; D010746:Phosphonoacetic Acid