Parathyroid hormone degradation by opossum kidney cells via receptor-mediated endocytosis and lysosomal hydrolysis

Abstract

The mechanisms involved in parathyroid hormone (PTH) degradation by proximal renal tubule cells were studied using an opossum kidney cell line possessing PTH receptors as an in vitro model system. One hour incubation of 5 nmol/l human (h) PTH-(1-84) with intact opossum kidney cells (4.0\m=x\ 106 cells) resulted in about 70% degradation and disappearance of hPTH-(1-84) from the medium, as determined by a two-site immunoradiometric assay. Preincubation with 100 nmol/l h[Nle8, Nle18, Tyr34]PTH-(1-34)amide for 6, 24, 48 and 72 h caused a 26, 47, 62 and 73% decrease, respectively, in PTH degradation by opossum kidney cells. Binding studies with 125I-labeled h[Nle8, Nle18, Tyr34]PTH-(1\x=req-\ 34)amide as a radioligand showed that PTH receptor binding decreased with the time of pretreatment with the agonist. Pretreatments of the cells with monensin, an inhibitor of endocytosis, and the lysosomotropic agents such as chloroquine, ammonium chloride and leupeptin, inhibited degradation of hPTH-(1-84) by 87, 71, 76 and 72%, respectively. Concentrations of 5 nmol/l hPTH-(39-84) and hPTH-(39-68), which are known not to bind to PTH receptors appreciably, were not degraded by opossum kidney cells during 1 h incubations. Thus intact, biologically active PTH, but not its inactive fragments, is degraded by opossum kidney cells, by receptor-mediated endocytosis and lysosomal hydrolysis. A mechanism resembling the peritubular uptake of intact PTH by perfused kidneys reported previously appears to play a main role in PTH metabolism by cultured renal cells.

Megalin Antagonizes Activation of the Parathyroid Hormone Receptor

“…In opossum kidney cells most PTH was subjected to endocytosis and lysosomal degradation, whereas a minor fraction of the hormone was metabolized by extracellular chymotrypsin-like endopeptidases (10,11,27). It has been suggested that in opossum kidney cells the PTH/PTHrP receptor is responsible for the endocytic uptake of the hormone.…”

“…Finally, cellular uptake and degradation of PTH was demonstrated directly in cultured proximal tubular cells. In these cells, the PTH/PTHrP receptor was suggested to mediate the uptake and degradation of the hormone (10,11). Whether this receptor is also responsible for systemic clearance of PTH or whether alternative uptake mechanisms exist in vivo remained unclear.…”

Enzymological and cellular mechanisms of parathyroid hormone degradation by the kidney

“…AI-ternatively, OK cells are suitable for studying PTH degradation at the basolateral surface specifically, because the effect of luminal microvillar hydrolytic activities is negligible. Indeed, our study showed that C-terminus or mid-portion of PTH, hPTH-(39-84) or hPTH-(39-68), which are known to be hydrolyzed solely at the luminal microvillar membranes in animal studies [1,6], were scarcely degraded by the monolayer cultures" of OK cells [26]. In contrast, about 70 % of hPTH-(1-84) added in the media was readily hydrolyzed by these cells via a receptor-mediated endocytosis and subsequent lysosomal hydrolysis at the basolateral surfaces.…”