Luminal and basolateral uptake and receptor binding of IGF-I in rabbit renal proximal tubules

The aim of the present study was to quantify and compare the luminal and basolateral binding and uptake of 125I-labeled insulin-like growth factor I (IGF-I) by means of 1) isolated, perfused, proximal tubules combined with electron microscope autoradiography and 2) luminal and basolateral membrane vesicles from rabbit proximal tubules. 125I-IGF-I was added to isolated perfused proximal tubules for 30 min in concentrations of 1.6-3.9 micrograms/l to either the perfusate or the bath. The luminal and basolateral uptake in 30 min averaged 447 and 410 fg/mm, respectively. About 20% of the luminally absorbed IGF-I was digested. Addition of excess unlabeled IGF-I (10(-7) M) to the bath produced complete inhibition of the basolateral binding/uptake, whereas no inhibition of the luminal uptake was seen. Electron microscope autoradiography showed that IGF-I after luminal endocytic uptake to a large extent was transported into lysosomes. After basolateral exposure the major portion of the grains was found over the basolateral cell membrane; however, a significant amount was located over endocytic vacuoles and lysosomes in both apical and basal parts of the cells. In both luminal and basolateral membrane vesicles, single-class, high-affinity binding sites for IGF-I were found with dissociation constants of 6.3 and 5.7 nM, respectively. Specific binding capacities averaged 2.7 and 25.7 pmol IGF-I/mg protein in luminal and basolateral vesicles. The biochemical data suggest an asymmetric distribution of specific IGF-I receptors in the luminal and basolateral membranes, with a greater abundance of receptors in the latter. The extensive basolateral endocytic binding/uptake of IGF-I compared with that of the luminal in isolated perfused tubules differs considerably from the processing of other peptide hormones.

Stepwise degradation of NT in pars convoluta and recta of rabbit proximal tubules: evidence of axial heterogeneity

Reabsorption and degradation of the neuropeptide neurotensin (NT) in rabbit proximal pars convoluta (PC) and pars recta (PR) nephron segments were characterized. Brush-border membrane vesicle fractions (PC or PR) were incubated with [3H]NT, and the extent and pattern of peptide hydrolysis were determined by reversed-phase high-pressure liquid chromatography (rHPLC). Furthermore, isolated rabbit PC and PR segments were perfused with [3H]NT, reabsorption of [3H]NT was quantified, and the collected perfusate was analyzed by HPLC. Metabolites were characterized. Finally, rabbit proximal tubules were microinfused in vivo with [3H]NT to follow the tubular uptake by electron microscope autoradiography. Degradation increased with time in both vesicle fractions. The main difference was an extensive cleavage of NT in PR, as revealed by a higher proportion of end metabolites. This was also visualized as a higher proportion of the large degradation product in rHPLC fraction 39 [NT-(1–11)] in PC as compared with PR after 30 min of incubation. The isolated perfused proximal tubular segments processed NT with large efficiency. PC segments processed 90% of the perfused amount, and PR processed 88%. Only 13% in PC and 10% in PR of the processed NT were found in the bath and the tubule. The main part of processed NT was in the collected perfusate, and rHLPC profiles revealed that NT-(1–11) was the only metabolite in both PC and PR. Electron microscope autoradiography demonstrated autoradiographic grains over invaginations and over the apical part of the proximal tubule cell in endocytic vesicles and vacuoles 10 min after microinfusion of [3H]NT.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide YY luminal processing and axial heterogeneity of basolateral binding in renal proximal tubules

This study investigates the definite location of peptide YY (PYY) binding sites on the basolateral membranes in proximal tubules. S1, S2, and S3 segments were dissected, perfused in vitro, and exposed to [125I-Tyr36]monoiodo-PYY either in the bath fluid or in the perfusate. S1 segments exposed to [125I-Tyr36]PYY in the bath fluid were fixed and prepared for electron microscope autoradiography. The results demonstrated a high degree of axial heterogeneity of basolateral binding of PYY, since only S1 bound PYY, 0.59 +/- 0.09 pg/mm after 15 min; 89.1% could be displaced with unlabeled PYY. PYY was not internalized, 90% of the grains were associated with the basolateral membranes, and no accumulation of grains was observed over the vacuolar apparatus. After luminal perfusion with PYY, 79.3 +/- 7.2% was processed, 61.7 +/- 6.3% was degraded at the brush border, and no tubular accumulation was detected. Thus PYY is not taken up by endocytosis. Unexpectedly, a very large fraction of processed PYY was transported from lumen to bath as trichloroacetic acid (TCA)-precipitable label constituting 41.6 +/- 4.7%. There was no axial heterogeneity in the luminal handling of PYY. In conclusion, this study reveals a high density of PYY binding sites at the basolateral membranes from S1 segments, indicating a selective function of S1 segments on stimulation with PYY. In contrast to other proteins PYY was not internalized from the basolateral membranes.