Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

Corticosterone binding sites along the rat nephron

1983 ◽  
Vol 244 (5) ◽  
pp. F504-F509 ◽  
Author(s):  
S. M. Lee ◽  
M. A. Chekal ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Glucocorticoid Receptors ◽  
Binding Capacity ◽  
Specific Binding ◽  
Thick Ascending Limb ◽  
Main Target ◽  
Collecting Tubule ◽  
Pars Recta ◽  
Kidney Functions ◽  
Cortical Collecting Tubule

Glucocorticoids influence numerous kidney functions but the precise location of glucocorticoid receptors in the nephron is not known. To identify the renal binding sites of corticosterone, the natural glucocorticoid in the rat, we measured the binding of [3H]corticosterone to discrete nephron segments microdissected from adrenalectomized rats. Highest specific binding capacity at 25 degrees C (expressed as fmol X cm-1 +/- SE) was found in the cortical collecting tubule (9.69 +/- 0.77) followed in decreasing order by the distal convoluted tubule (2.70 +/- 0.49), medullary collecting tubule (2.58 +/- 0.64), proximal convoluted tubule (1.09 +/- 0.10), and pars recta (0.57 +/- 0.08). Binding was lowest in the thick ascending limb of Henle's loop, with comparable values in the medullary (0.27 +/- 0.05) and cortical (0.26 +/- 0.05) portions of this segment. The apparent maximal binding capacity of the cortical collecting tubule for corticosterone exceeded by nearly two orders of magnitude that of aldosterone previously measured by us in this structure, which is in agreement with the observations of other investigators in kidney cytosol. Specific binding of corticosterone can be demonstrated along the entire rat nephron, but binding sites are concentrated in the cortical collecting tubule. This segment appears to be the main target site for corticosterone as it is for aldosterone.

scholarly journals Influence of divalent cations on the detection of somatogenic and lactogenic binding sites in mouse liver cells

1985 ◽  
Vol 228 (3) ◽  
pp. 761-764 ◽  
Author(s):  
G N Ciccia-Torres ◽  
J M Dellacha
Keyword(s):  
Binding Sites ◽  
Mouse Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Divalent Cations ◽  
Isolated Hepatocytes ◽  
Liver Cells ◽  
Scatchard Analysis ◽  
Male Mice ◽  
Ovine Prolactin

Specific binding of 125I-labelled human somatotropin was demonstrated in isolated hepatocytes from male mice. In the presence of divalent cations (Ca2+ and Mg2+) the binding of 125I-labelled human somatotropin was competitive with ovine prolactin. Scatchard analysis of competition data indicated a KD of 1.4 +/- 0.2 nM and a binding capacity of 13 000 +/- 2000 sites/cell. In the absence of divalent cations and in the presence of EDTA, human and bovine somatotropins were found to be equally effective to displace bound 125I-labelled human somatotropin, while ovine prolactin showed a weak competition. In this case, the binding capacity was 8400 +/- 1500 sites/cell and the KD was 1.1 +/- 0.1 nM.

Benzodiazepines inhibit transport-related oxygen consumption in thick ascending limb

1988 ◽  
Vol 255 (3) ◽  
pp. C385-C392 ◽  
Author(s):  
F. N. Ziyadeh ◽  
Z. S. Agus
Keyword(s):  
Oxygen Consumption ◽  
Amphotericin B ◽  
Binding Sites ◽  
Cell Function ◽  
Chloride Transport ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Thick Ascending Limb ◽  
Total Oxygen ◽  
Sodium Uptake

Specific binding sites for benzodiazepines (BZD) have been identified in several nonneuronal tissues including the kidney where they are localized predominantly to the tubular epithelium of the thick ascending limb of Henle's loop (TALH). The physiological function of these nonneuronal (peripheral) BZD-binding sites is undefined, but it has been suggested that they may represent receptors for putative endogenous ligands that may modulate cell function. In the current study, we examined the in vitro effects of diazepam and Ro5-4864, a specific peripheral BZD-receptor agonist, on the oxygen consumption of medullary TALH tubule suspensions of rabbit kidney. Maximal inhibition of total oxygen consumption was achieved at a dose of 5 X 10(-4) M of either agent. On average, diazepam and Ro5-4864 reduced total oxygen consumption by 41 and 44%, respectively. The predominant inhibition was in the ouabain-sensitive component of oxygen consumption, which suggests that BZDs inhibit active sodium-chloride transport in the TALH. To assess whether this inhibition depends on sodium uptake, TALH tubules were pretreated with amphotericin B (2 X 10(-6) M) to enhance sodium uptake and stimulate basal oxygen consumption; subsequent addition of Ro5-4864 (5 X 10(-4) M) still reduced oxygen consumption to a residual value that was not different from that in TALH tubules treated with Ro5-4864 but without pretreatment with amphotericin B. This suggests that BZD inhibition of transport-related oxygen consumption is not caused by diminution of sodium uptake into cells and thus appears to be distinct from the effect of furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

PGF2 alpha and PGE2 binding to rat myometrium during gestation, parturition, and postpartum

1990 ◽  
Vol 258 (5) ◽  
pp. E740-E747
Author(s):  
M. Molnar ◽  
F. Hertelendy
Keyword(s):  
Placebo Group ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Time Dependent ◽  
Scatchard Analysis ◽  
Plasma Progesterone ◽  
High Affinity ◽  
The Third ◽  
Venous Plasma

The specific binding of prostaglandins (PG) F2 alpha and E2 was studied in a rat myometrial membrane-enriched fraction during the latter part of gestation and parturition, as well as in the postpartal period. Tritiated PGE2 and PGF2 alpha binding was specific, saturable, time dependent, and directly proportional to the amount of membrane protein. Scatchard analysis indicated the presence of high-affinity (Kd2) and low-affinity (Kd2) binding sites for both PGs. The affinity of both binding sites for PGF2 alpha and the apparent Kd2 for PGE2 remained essentially the same throughout gestation and post-partially and were similar to nonpregnant rats. The apparent Kd1 of PGE2, however, increased by 10-fold from day 21 of gestation to 1 day postpartum. Although the maximal binding capacity of the high-affinity (Bmax1) and low-affinity (Bmax2) binding sites of PGF2 alpha showed a nonsignificant increase compared with prepartum values, reaching maximal values 12-24 h postpartum, those of PGE2 showed a significant increase on the third day after delivery. The concentration of prostanoids in uterine venous plasma and amniotic fluid increased significantly with approaching parturition, whereas plasma progesterone decreased, raising the estradiol-progesterone ratio 25-fold. After unilateral fetectomy, the binding sites for PGF2 alpha and PGE2 increased significantly compared with the contralateral pregnant horns. Administration of the PG synthetase inhibitor, indomethacin, also increased two- to threefold both PGF2 alpha and PGE2 binding compared with the placebo group, whereas intrauterine administration of PGF2 alpha and PGE2 significantly reduced it.(ABSTRACT TRUNCATED AT 250 WORDS)

INHIBITORY EFFECTS OF ITP SERA ON BINDING OF ANTIPLATELET GLYCOPROTEIN (GP) IIb/IIIa MONOCLONAL ANTIBODIES TO HUMAN UMBILICAL VASCULAR ENDOTHELIAL CELLS (HUVE)

1987 ◽  
Author(s):  
T Nakajima ◽  
T Koyama ◽  
Y Nishida ◽  
H Tanaka ◽  
E Kakishita ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Vascular Endothelial Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Cell Pellet ◽  
A Cell ◽  
Molecular Masses ◽  
The Difference

Some ITP patients have specific autoantibodies to platelet GP IIb/IIIa. On the other hand, HUVE were shown to synthesize platelet GP IIb/IIIa like substances. Therefore, we studied the binding of ITP sera to HUVE by showing the inhibitory effect of ITP sera on the binding of anti-platelet GP IIb/IIIa monoclonal antibodies to HUVE. HUVE were cultured according to the method of Jaffe et al. 125-I-anti-platelet GP IIb/IIIa monoclonal antibody (125-I-Anti-GP) (40.3 mCi/mg), 40 yl, was added to a cell suspension of HUVE (1.5 × 104/500 μl) in a plastic RIA tube. After incubation for 30 min. at 4°C and centrifugation of 10,000 xg for 3 min., the radioactivity of the cell pellet was measured. Specific binding was determined by determining the difference between cell-bound radioactivity in the absence and presence of an excess amount of unlabelled ligand at 100 x concentrations. Scatchard analysis using 125-I-Anti-GP showed that the maximum binding capacity was 8 × 104/cell and Kd was 40.2 nM. The binding rate of 125-I-Anti-GP to HUVE treated with ITP (high PAIgG) sera (n=6) was 15.2±3.3% compared with 24.0±7.5%, observed for HUVE treated with normal sera (n=10). Treatment of ITP sera to HUVE significantly lowered the binding of 125-I-Anti-GP to HUVE (P<0.05). A combined analysis of SDS-PAGE and Western blotting of washed platelet and endothelial cell lysates shows that two proteins from each cells had similar or identical molecular masses to GP IIb/IIIa.These findings show that there are GP IIb/IIIa on the HUVE, ITP sera from our patients may have antibodies to HUVE GP IIb/IIIa and that anti-platelet GP IIb/IIIa antibodies in the ITP sera may bound not only to some platelets, but also to the HUVE

scholarly journals Human somatotropin binding to rabbit kidney microsomal fraction

1981 ◽  
Vol 200 (2) ◽  
pp. 257-264 ◽  
Author(s):  
L P Roguin ◽  
S H Sánchez ◽  
J S Bonifacino ◽  
A C Paladini
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Binding Activity ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Temperature Dependent ◽  
Binding Reaction ◽  
Dissociation Equilibrium ◽  
Microsomal Membranes ◽  
Kidney Microsomes

Specific binding of 125I-labelled human somatotropin was demonstrated in microsomal membranes (microsomes) from rat and rabbit kidneys. Female rabbit kidney microsomes showed the highest binding activity and were used for further study. The association of 125I-labelled human somatotropin was time- and temperature-dependent and the binding reaction was reversible. Scatchard analysis of saturation data indicated a dissociation equilibrium constant, KD, of 56 pM and a binding capacity of 37 fmol per mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the microsomes was specifically inhibited by hormones with lactogenic activity. The binding sites, as well as 125I-labelled human somatotropin, were not inactivated on incubation. Treatment of the microsomes with trypsin and chymotrypsin decreased the specific binding by over 90%. Preheating of the microsomes at 55 degrees C for 15 min abolished 50% of the specific binding activity.

scholarly journals Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver

1984 ◽  
Vol 219 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
Y A Lefebvre ◽  
J T Venkatraman
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Intact Animal ◽  
Binding Capacity ◽  
Specific Binding ◽  
Male Rat ◽  
Scatchard Analysis ◽  
Specific Binding Sites ◽  
Number Of Binding Sites

Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3′,5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3′,5′-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites.

Binding sites for interferons on ovine and human endometrial membranes

1993 ◽  
Vol 5 (2) ◽  
pp. 219 ◽  
Author(s):  
DL Russell ◽  
GG Manalo ◽  
JK Findlay ◽  
LA Salamonsen
Keyword(s):  
Binding Sites ◽  
Luteal Phase ◽  
Binding Capacity ◽  
Specific Binding ◽  
Major Product ◽  
Human Endometrium ◽  
Scatchard Analysis ◽  
Ovarian Steroids ◽  
Steroid Dependence ◽  
Proliferative Phase

In the ewe, the major product of the preimplantation blastocyst is ovine trophoblast protein-1 (oTP-1), which is now classified as an omega-interferon (IFN). Receptors for IFN are present on sheep endometrium and vary cyclically, presumably modified by the actions of ovarian steroids. This study examined whether or not IFN receptors were present on human endometrium at any stage during the menstrual cycle. In addition, the steroid dependence of ovine endometrial IFN receptors was determined. Specific binding of 125I-labelled IFN (125I-IFN) to ovine endometrial membranes was substantially higher than binding to membranes derived from bovine spleen, human placenta or pooled human endometrium (relative specific binding 100:33:36:20). Human endometrial membrane preparations from proliferative-phase tissue showed very little specific binding (mean 0.8 +/- 0.3%, n = 4) in contrast to luteal-phase endometrium (2.1 +/- 0.3%, n = 8). Treatment of ovariectomized ewes with oestradiol-17 beta (E) resulted in significantly increased binding (117 +/- 7%) of 125I-IFN to endometrial tissues compared with tissue from ovariectomized (OvX, 75 +/- 7%), progesterone (P)-treated (69 +/- 7%), or (E + P)-treated (81 +/- 8%) groups (P < 0.05); all were compared with binding to pooled ovine luteal-phase tissue, 100%. There were no differences between the other three groups. Scatchard analysis showed binding affinity of the same order for the sheep and human receptors (Kd = 10(-10) mol L-1) but binding capacity was considerably lower for human (6.0 fmol mg-1) than for sheep (47-123 fmol mg-1) endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell culture of renal epithelium derived from rabbit microdissected cortical collecting tubules

1983 ◽  
Vol 244 (6) ◽  
pp. F724-F728 ◽  
Author(s):  
M. G. Currie ◽  
B. R. Cole ◽  
K. DeSchryver-Kecskemeti ◽  
S. Holmberg ◽  
P. Needleman
Keyword(s):  
Biochemical Characterization ◽  
Cultured Cells ◽  
Fold Increase ◽  
Cell Types ◽  
Rabbit Kidney ◽  
Renal Epithelium ◽  
Distinct Cell ◽  
Collecting Tubule ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

Cortical collecting tubules were dissected from rabbit kidney and cultured in a hormonally defined serum-free medium. Morphologic studies of the cultured cells derived from the collecting tubule indicated that the cells maintained their epithelial nature. These studies also revealed the presence of two distinct cell types that closely resemble the principal and intercalated cell types of the cortical collecting tubule. Several biochemical characteristics of the cultured cells were found to be similar to previously reported values for the cortical collecting tubule. The cells retain hormonal responsiveness to antidiuretic hormone (ADH), as demonstrated by a 12-fold increase in cAMP in response to ADH. Cultured cortical collecting tubule cells produce prostaglandins, with prostaglandin E2 as the predominant cyclooxygenase product. This study presents the first morphologic and biochemical characterization of cortical collecting tubule epithelial cells grown in culture.

Segmental synthesis and actions of prostaglandins along the nephron

1987 ◽  
Vol 253 (3) ◽  
pp. F377-F387 ◽  
Author(s):  
J. P. Bonvalet ◽  
P. Pradelles ◽  
N. Farman
Keyword(s):  
Renal Tubule ◽  
Thick Ascending Limb ◽  
Pge2 Synthesis ◽  
Complex Interactions ◽  
Quantitative Measurements ◽  
Medullary Thick Ascending Limb ◽  
Exogenous Substrate ◽  
Collecting Tubule ◽  
The Difference ◽  
Cortical Collecting Tubule

The sites of synthesis and action of prostaglandins (PGs) along the renal tubule are examined. We focused our attention on experiments performed on well-defined nephron segments, using direct quantitative measurements of prostaglandin synthesis by radio- or enzyme-immunoassay. On the other hand, we selected, among the described effects of PGs, those obtained on precisely defined tubular segments. Among PGs, PGE2 synthesis is largely predominant all along the tubule. Its main sites of synthesis are the medullary collecting tubule and, to a lesser extent, the cortical collecting tubule and the thin limb of Henle's loop. Synthesis of PGE2 is amplified approximately tenfold in the presence of an excess exogenous substrate, arachidonic acid, compared with values measured without addition of substrate. Other eicosanoids have roughly the same distribution along the tubule as PGE2. Their rate of synthesis is, however, much less than that of PGE2, approximately 20-fold lower for PGF2 alpha and 6-keto-PGF1 alpha, and 100-fold lower for thromboxane B2 (TxB2). This contrasts with glomerular PG synthesis, where the difference between the production of PGE2 and other eicosanoids is much less marked. Most studies agree that antidiuretic hormone (ADH) and kinins augment PGE2 synthesis, whereas corticosteroids decrease it, at least in the collecting tubule. Direct effects of PGE2 have been described mainly in the medullary thick ascending limb and collecting tubule. They generally consist of a decrease in transepithelial potential difference and reabsorptive rates of water and solutes, in particular sodium and chloride. However, whatever the solute or tubular segment concerned, some studies failed to find such effects. The bulk of evidence suggests that ADH and PGs interact in kidney tubular cells. It is generally accepted that PGs antagonize the hydrosmotic effects of ADH in the collecting tubule. The mechanisms underlying these complex interactions are still under discussion: they probably involve several types of receptors and pathways for ADH action, which intervene in the modulation of both PG synthesis and cyclic nucleotides, and several types of PG receptors, either stimulatory or inhibitory to adenylate cyclase.

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