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)

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.

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.

Incorporation of Cholesterol into Peripheral Nerve Myelin

1972 ◽  
Vol 30 ◽  
pp. 334-335
Author(s):  
Frank A. Rawlins
Keyword(s):  
Electron Microscope ◽  
Sciatic Nerve ◽  
Electron Microscope Autoradiography ◽  
Myelin Sheaths ◽  
Microscope Autoradiography ◽  
Grain Distribution ◽  
Sciatic Nerves ◽  
Autoradiographic Analysis ◽  
Old Mice ◽  
Short Times

Several speculations exist as to the site of incorporation of preformed molecules into myelin. The possibility that an autoradiographic analysis of cholesterol-1,2-H3 incorporation at very short times after injection might shed some light in the solution of that problem led to the present experiment.Cholesterol-1,2-H3 was injected intraperitoneally into 24 tenday old mice. The animals were then sacrificed at 10,20,30,40,60,90,120 and 180 min after the injection and the sciatic nerves were processed for electron microscope autoradiography. To analyze the grain distribution in the autoradiograms of cross and longitudinal sections from each sciatic nerve myelin sheaths were subdivided into three compartments named: outer 1/3, middle 1/3 and inner 1/3 compartments.It was found that twenty min. after the injection of cholesterol -1.2-H3 (Figs. 1 and 2), 55% of the total number of grains (t.n.g) found in myelin were within the outer 1/3 compartment, 9% were within the middle 1/3 and 36% within the inner 1/3 compartment

Depression of calcium pump activity in renal cortex of vitamin D-deficient rats with secondary hyperparathyroidism

1990 ◽  
Vol 123 (4) ◽  
pp. 438-444 ◽  
Author(s):  
Yusuke Tsukamoto ◽  
Teiichi Tamura ◽  
Michiyo Saitoh ◽  
Yumiko Takita ◽  
Toshiaki Nakano
Keyword(s):  
Vitamin D ◽  
Secondary Hyperparathyroidism ◽  
Hormonal Regulation ◽  
Serum Calcium Level ◽  
Renal Cortex ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Basolateral Membrane Vesicles ◽  
Pump Activity

Abstract. To examine the hormonal regulation of the ATP-dependent Ca2+ pump in the kidneys, the ATP-dependent Ca2+ uptake by the basolateral membrane vesicles in the renal cortex was measured using radioactive calcium (45Ca2+) in rats with vitamin D deficiency or rats undergoing thyroparathyroidectomy. The Vmax of the Ca2+ pump activity was increased not only by administering calcitriol, but also by normalizing the serum calcium level in vitamin D-deficient rats. PTH suppressed the Ca2+ pump activity in normocalcemic vitamin D-deficient rats. Thyroparathyroidectomy did not affect the Ca2+ pump activity in the kidneys of normal rats. It was concluded that the ATP-dependent Ca2+ pump activity was depressed by secondary hyperparathyroidism in vitamin D-deficient rats.

scholarly journals Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex

1989 ◽  
Vol 264 (1) ◽  
pp. 223-231 ◽  
Author(s):  
T C Williams ◽  
A J Doherty ◽  
D A Griffith ◽  
S M Jarvis
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Facilitated Diffusion ◽  
Basolateral Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Outer Cortex ◽  
Overshoot Phenomenon

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

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