Stimulation of calcium uptake by parathyroid hormone in renal brush-border membrane vesicles. Relationship to membrane phosphorylation

The effect of parathyroid hormone (PTH) on Ca²⁺ uptake was studied in brush-border membrane vesicles (BBMV) prepared from the kidneys of dogs administered 4-5 μg/kg of bovine PTH 1-84 in vivo. PTH stimulated Ca²⁺ uptake at 20 s of incubation from control values of 231 ± 21 to 306 ± 30 pmol/mg of protein, p<0.001. The stimulation of Ca²⁺ uptake by PTH was not reversed by incubation of the BBMV with the Ca²⁺ ionophore, despite the fact that Ca²⁺ uptake was several times greater than the expected uptake at equilibrium, indicating that most of the uptake represented Ca²⁺ binding to the BBMV. In BBMV from kidneys exposed to PTH, hypotonic lysis or increasing the osmolality of the solution external to the BBMV did not affect Ca²⁺ uptake. These data also indicated that the largest fraction of Ca²⁺ uptake in the presence of a chemical potential represented binding of Ca²⁺ to BBMV. Ca²⁺ binding was initially to the exterior of the BBMV, then translocated within the membrane and to the interior vesicular face as assessed by chelation of Ca²⁺ bound to the BBMV by ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Incubation of BBMV from kidneys exposed to PTH with gentamicin, which competes with Ca²⁺ for anionic phospholipid-binding sites, reversed the stimulatory effects of PTH on Ca²⁺ uptake. Phosphorylation of BBMV and PTH treatment in vivo had similar effects on BBMV phospholipid composition increasing the levels of anionic phospholipids. Phosphorylation of the BBMV also produced gentamicin-inhibitable increases in membrane Ca²⁺ binding. Phosphorylation of the BBMV from kidneys exposed to PTH was inhibited suggesting a higher state of phosphorylation in vivo. The data demonstrate that PTH administered in vivo stimulated Ca²⁺ binding in BBMV that was gentamicin inhibitable and associated with an increase in the membrane content of anionic phospholipids.