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

1991 ◽  
Vol 260 (3) ◽  
pp. F359-F367
Author(s):  
S. Nielsen ◽  
S. P. Sheikh ◽  
M. I. Sheikh ◽  
E. I. Christensen
Keyword(s):  
Binding Sites ◽  
Peptide Yy ◽  
Large Fraction ◽  
Proximal Tubules ◽  
Electron Microscope Autoradiography ◽  
Basolateral Membranes ◽  
Microscope Autoradiography ◽  
Luminal Perfusion ◽  
High Degree

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.

ABSORPTION OF 125I-LABELLED SHEEP GROWTH HORMONE IN SINGLE PROXIMAL TUBULES OF THE RAT KIDNEY

1976 ◽  
Vol 68 (1) ◽  
pp. 21-NP ◽  
Author(s):  
B. D. STACY ◽  
A. L. C. WALLACE ◽  
R. T. GEMMELL ◽  
B. W. WILSON
Keyword(s):  
Growth Hormone ◽  
Renal Cortex ◽  
Rat Kidney ◽  
Alternative Pathway ◽  
Proximal Tubules ◽  
Lysosomal Degradation ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Kidney Micropuncture

SUMMARY Techniques of kidney micropuncture and electron microscope autoradiography have been used to study the uptake of 125I-labelled sheep growth hormone (GH) in rat renal proximal tubules. After microperfusion of a proximal tubule with 125I-labelled GH, the transport of label by the tubular epithelium was studied autoradiographically at selected times up to 1 h. The sequential transfer of labelled material from tubule to microvilli, then to small and large apical vacuoles and finally to lysosomes followed the pattern of absorption that has been described for other proteins. Evidence of lysosomal degradation of the transported protein was obtained from studies in vitro; lysosomes isolated from the renal cortex rapidly converted 125I-labelled GH to products of lower molecular weight. In addition to the absorptive pathway through the intracellular vacuolar apparatus it appeared that there was also an alternative pathway, less well defined, whereby GH could be absorbed without being degraded.

Luminal and basolateral uptake of insulin in isolated, perfused, proximal tubules

1987 ◽  
Vol 253 (5) ◽  
pp. F857-F867 ◽  
Author(s):  
S. Nielsen ◽  
J. T. Nielsen ◽  
E. I. Christensen
Keyword(s):  
Electron Microscope ◽  
Significant Inhibition ◽  
Proximal Tubules ◽  
Specific Mechanism ◽  
Electron Microscope Autoradiography ◽  
Basolateral Membranes ◽  
No Inhibition ◽  
Microscope Autoradiography ◽  
High Insulin ◽  
High Concentrations

The present study was performed to quantitate and compare the luminal and the peritubular uptake of 125I-labeled insulin in isolated, perfused, proximal tubules from rabbit kidneys. 125I-insulin was added in physiological concentrations of 3.0-7.0 ng/ml or 59.0-89.5 ng/ml (high insulin concentrations) to either the perfusate or the bath fluid for 30 min. The luminal uptake in 30 min averaged 0.76 pg/mm at physiological concentrations and 18.0 pg/mm at high insulin concentrations. About 15-41% of the absorbed insulin was digested and less than 5% was transported from the lumen to the peritubular space as intact insulin. The peritubular binding/uptake of 125I-insulin at physiological and high concentrations in the bath was 0.136 and 0.318 pg, respectively. Addition of excess unlabeled insulin (10(-5) M) to the bath produced significant inhibition of binding (53.7%) at 7.0 ng/ml, but no inhibition at 89.5 ng/ml labeled insulin in the bath. This indicates that insulin is bound/absorbed at the basolateral membranes both by a saturable specific mechanism and a nonspecific, nonsaturable mechanism. The basolateral absorption constituted 15.2 and 1.8% of the total tubular extraction of insulin at physiological and high insulin concentrations, respectively. Electron microscope autoradiography showed that, after luminal as well as basolateral endocytosis, insulin was exclusively accumulated in endocytic vacuoles and lysosomes.

Synthesis and Localization of Sulphated Mucopolysaccharide in Megakaryocytes and Platelets of the Rat, An Analysis by Electron-Microscope Autoradiography

1972 ◽  
Vol 10 (3) ◽  
pp. 705-717
Author(s):  
G. G. MacPHERSON
Keyword(s):  
Electron Microscope ◽  
Golgi Apparatus ◽  
Blood Platelets ◽  
Total Activity ◽  
Electron Microscope Autoradiography ◽  
Dense Granules ◽  
Level Of Activity ◽  
Microscope Autoradiography ◽  
Almost All

Electron-microscope autoradiography has been used to investigate the synthesis and localization of sulphated mucopolysaccharide in megakaryocytes and blood platelets. Following 10-min incubation of bone marrow with 35S-sulpahte in vitro the majority of the activity in megakaryocytes was associated with the Golgi apparatus, but a substantial proportion was associated with other cytoplasmic organelles, suggesting either rapid transport or sulphation of mucopolysaccharide outside the Golgi apparatus. Three hours after the intravenous injection of 35SO4 only a small proportion of the total activity was associated with the Golgi apparatus, most being associated with demarcation membranes and dense granules, while 12 h after injection almost all the activity was associated with demarcation membranes and granules. A rising proportion of activity localized solely on the demarcation membranes suggested that they may possess some activity of their own. Autoradiographs of blood platelets prepared 72 h after the injection of 35SO4 were analysed. It was shown that most of the activity was associated with the α-granules, but there was strong evidence that the platelet membrane possessed a low level of activity.

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

1993 ◽  
Vol 265 (5) ◽  
pp. F624-F633 ◽  
Author(s):  
A. Flyvbjerg ◽  
S. Nielsen ◽  
M. I. Sheikh ◽  
C. Jacobsen ◽  
H. Orskov ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Dissociation Constants ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Proximal Tubules ◽  
Asymmetric Distribution ◽  
Basolateral Membrane Vesicles ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Igf I

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

1993 ◽  
Vol 264 (1) ◽  
pp. E45-E53
Author(s):  
T. Bjerke ◽  
S. Nielsen ◽  
M. I. Sheikh ◽  
E. I. Christensen
Keyword(s):  
Electron Microscope ◽  
Membrane Vesicle ◽  
Reversed Phase ◽  
Proximal Tubules ◽  
Proximal Tubule Cell ◽  
Peptide Hydrolysis ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Pars Convoluta

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)

Electron microscope autoradiography of isolated molecules

1978 ◽  
Vol 36 (2) ◽  
pp. 192-193
Author(s):  
M. Bouteille ◽  
E. Delain ◽  
N. Angelier
Keyword(s):  
Electron Microscope ◽  
High Efficiency ◽  
Specific Activity ◽  
Molecular Complexes ◽  
Electron Microscope Autoradiography ◽  
Microscope Autoradiography ◽  
Loop Technique ◽  
Silver Grains ◽  
Isolated Molecules

The LIGOP method of electron microscope autoradiography which consists in a combination of coating Ilford emulsion with the loop technique and developing with gold latensification and phenidon has proved to provide small, compact developed silver grains with high efficiency.This has made it possible to use this technique with very small materials such as isolated molecules of molecular complexes.The method was assayed first with 3H-Thymidine labelled T7 phages DNA molecule with 630,000 cpm/μg specific activity (fig. 1). The molecules were spread using the adsorption technique constrasted by rotatory shadowing with platinum and then subjected to autoradiography. The Labelling was sufficient to obtain quantitative data in which the spread molecules were considered as a material comparable to a “hot line”. The efficiency (45%) and the HD value (1600 Å) were calculated.The method was also applied to transcription units of pleurodeles oocytes nucleoli (fig. 2) labelled in vitro with 3H-Uridine.

scholarly journals Nature and characteristics of the binding of oestradiol-17β to a uterine macromolecular fraction

1968 ◽  
Vol 107 (6) ◽  
pp. 765-774 ◽  
Author(s):  
G. P. Talwar ◽  
M. L. Sopori ◽  
D. K. Biswas ◽  
S. J. Segal
Keyword(s):  
Serum Albumin ◽  
Binding Sites ◽  
Physiological Function ◽  
Receptor Protein ◽  
Binding Properties ◽  
Ph Values ◽  
Binding Function ◽  
High Degree ◽  
Protein Moiety

The binding of oestradiol-17β to two proteins, namely serum albumin and a uterus fraction, was studied in vitro. The former protein has a physiological function in the transport of the hormone and the latter is involved in the selective uptake of the steroid by the target organ. The uterus fraction shows a high degree of stereospecificity for the binding of the steroid. Cortisone, oestradiol-17α and testosterone are bound negligibly and progesterone to a much smaller extent than is oestradiol-17β. This property is in contrast with the wide variety of ligands bound by the serum albumin. The temperature and the presence of the steroid influence markedly the binding properties. Oestradiol binding to the uterus fraction is optimum at 37° and at pH7–8·5. It is markedly decreased at pH values above or below this range, suggesting stringent conformational requirements. The tissue ‘receptor’ protein is a macromolecule with a minimum molecular weight of 100000. The protein moiety is essential for the binding function. The probable concentration of the total binding sites for oestradiol in the ovariectomized-rat uterus cytoplasmic fraction as determined in vitro is about 1mμm at a steroid concentration of 50mμm.

scholarly journals Cytological events involved in protein synthesis in cellular and syncytial trophoblast of human placenta. An electron microscope autoradiographic study of [3H]leucine incorporation.

1978 ◽  
Vol 76 (2) ◽  
pp. 400-417 ◽  
Author(s):  
D M Nelson ◽  
A C Enders ◽  
B F King
Keyword(s):  
Protein Synthesis ◽  
Electron Microscope ◽  
Autoradiographic Study ◽  
Leucine Incorporation ◽  
Electron Microscope Autoradiography ◽  
Placental Villi ◽  
Microscope Autoradiography ◽  
Syncytial Trophoblast ◽  
Time Point

Electron microscope autoradiography has been used to study protein synthesis in syncytial and cellular trophoblast of term human placental villi incubated in vitro with tritiated leucine ([3H]leu). Autoradiographs were analyzed using the hypothetical grain analysis of Blackett and Parry (1973. J. Cell Biol. 57:9-15). The results of this study demonstrated that both cellular and syncytial trophoblast have marked capacities for protein synthesis. Cellular trophoblast synthesized protein in both its rough endoplasmic reticulum (RER) and its ground plasm which contained abundant free ribosomes. The vast majority of 3H-proteins remained within the cell, with some of the proteins synthesized ultimately appearing in the nucleus. A small percentage of grains was ultimately associated with the trophoblast basement membrane. In syncytial trophoblast, the RER was the dominant site for protein synthesis. The autoradiographic data suggested that, as in the cellular trophoblast, the vast majority of 3H-proteins synthesized by the syncytial trophoblast remained within the syncytial trophoblast throughout the incubation period. The major portion of [3H]leu-labeling present in the syncytial trophoblast of villi incubated the longest times (4 h+) remained in association with the RER. Labeled proteins did not become concentrated in syncytial trophoblast Golgi apparatus, vesicles, or granules. In contrast to cellular trophoblast, the nuclei in the syncytium did not contain 3H-proteins at any time-point studied.

Handling of lysozyme in isolated perfused proximal tubules

1986 ◽  
Vol 251 (5) ◽  
pp. F822-F830 ◽  
Author(s):  
J. T. Nielsen ◽  
S. Nielsen ◽  
E. I. Christensen
Keyword(s):  
Electron Microscope ◽  
Proximal Tubules ◽  
Electron Microscope Autoradiography ◽  
Transcellular Transport ◽  
Microscope Autoradiography ◽  
Grain Distribution ◽  
Autoradiographic Analysis

Isolated, perfused proximal tubules from rabbit were used to study the luminal endocytic uptake, digestion, and transcellular transport of 125I-lysozyme. Ten tubules were perfused for 20 min with 125I-lysozyme and [14C]inulin and then with tracer-free perfusate for additional 40 min before fixation. The uptake and digestion of lysozyme was calculated per millimeter tubule length. The transfer of intact lysozyme from perfusate to the bath was measured and compared with the transfer of inulin. Five tubules were processed for electron microscope autoradiography, and the grain distribution was analyzed quantitatively. The results show that 2.7% of the perfused amount of lysozyme was taken up, and 21.3% of the absorbed protein was digested. The present experiments demonstrate that the transfer of intact lysozyme from lumen to bath is not significantly different from the transfer of inulin. The autoradiographic analysis showed that lysozyme was localized mainly in endocytic vacuoles and lysosomes after 60 min of perfusion.

Degradation and transport of AVP by proximal tubule

1987 ◽  
Vol 253 (6) ◽  
pp. F1120-F1128
Author(s):  
F. A. Carone ◽  
E. I. Christensen ◽  
G. Flouret
Keyword(s):  
Proximal Tubule ◽  
High Performance ◽  
Reduced Glutathione ◽  
Proximal Tubules ◽  
Electron Microscope Autoradiography ◽  
Lysosomal Fraction ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

High-performance liquid chromatography (HPLC) analysis revealed that [3,4,5-3H-Phe3,Arg8]vasopressin ([3H]AVP) was not degraded by isolated renal brush-border membranes or by a cortical lysosomal fraction in vitro; however, in the presence of 1 mM reduced glutathione, [3H]AVP was degraded by both preparations. Renal cortical homogenates in vitro and luminal peptidases of proximal tubule in vivo degraded [3H]AVP and in both instances yielded phenylalanine, hexapeptide AVP 1-6, heptapeptide AVP 1-7, octapeptide AVP 1-8, and two uncharacterized products X and Y. These data suggest that filtered AVP is reduced in the proximal tubule by a reduced glutathione-dependent transhydrogenase and subsequently cleaved to [3H]Phe by tubular aminopeptidases. Following microinfusion of [3H]AVP into proximal tubules, 15.7% of the label was absorbed. Five and fifteen minutes after infusion of [3H]AVP, sequestration of total label in proximal tubules was 4.5 and 2.1%, respectively, and quantitative electron microscope autoradiography revealed accumulation of grains over apical endocytic vacuoles and lysosomes consistent with endocytic uptake and rapid lysosomal degradation of AVP and/or a large metabolite. Thus, enzymatic cleavage of AVP by luminal and lysosomal peptidases in proximal tubules could involve disulfide bond, C-terminal, and N-terminal loci.

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