Corticosteroid-binding studies in cytosol of colonic mucosa of the rat

1981 ◽  
Vol 240 (6) ◽  
pp. G417-G423
Author(s):  
E. T. Marusic ◽  
J. P. Hayslett ◽  
H. J. Binder
Keyword(s):  
Colonic Mucosa ◽  
Steroid Receptors ◽  
Binding Capacity ◽  
Potential Difference ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Minimal Dose ◽  
High Affinity Receptor ◽  
Affinity Receptor ◽  
Cytosolic Receptor

Cytosolic binding of [3H]dexamethasone was studied in the colon of the rat. [3H]dexamethasone binding was rapid and stable at 4 degrees C for 240 min. Scatchard analysis revealed a Kd of 6.2 +/- 0.5 X 10(-9) M and a binding capacity of 149 +/- 6.4 fmol/mg cytosolic protein (100,000-g fraction). Although dexamethasone inhibited [3H]dexamethasone binding more than that of aldosterone, [3H]aldosterone binding was inhibited equally by both aldosterone and dexamethasone. The relative order of potency of other steroids to inhibit [3H]dexamethasone binding was: dexamethasone greaterthan progesterone greater than spironolactone greater than aldosterone greater than corticosterone greater than cortexolone greater than estradiol. In other experiments, low doses of dexamethasone and aldosterone were infused into adrenalectomized animals to determine functional importance of these cytosolic steroid receptors. One-hour infusion of aldosterone at 2 micrograms/100 g body wt, which was the minimal dose of dexamethasone that increased transmural potential difference, did not alter the potential difference. These studies demonstrate the presence of a cytosolic receptor for dexamethasone and suggest that the action of dexamethasone on electrolyte transport in adrenalectomized animals is not mediated by the mineralocorticoid type of receptor and may be mediated by the specific high-affinity receptor for dexamethasone.

ATP-MgCl2 treatment after trauma-hemorrhage/resuscitation increases hepatocyte P2-purinoceptor binding capacity

1994 ◽  
Vol 266 (6) ◽  
pp. R1810-R1815
Author(s):  
M. S. Mahmoud ◽  
P. Wang ◽  
S. R. Hootman ◽  
S. S. Reich ◽  
I. H. Chaudry
Keyword(s):  
Binding Capacity ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Beneficial Effects ◽  
High Affinity ◽  
Receptor Component ◽  
Receptor Dynamics ◽  
Maximum Binding Capacity ◽  
High Affinity Receptor ◽  
Affinity Receptor

Although our studies indicate that P2-purinoceptor binding capacity decreases after hemorrhage and resuscitation, it is not known whether ATP-MgCl2 administration after hemorrhage has any beneficial effects on the receptor dynamics. To study this, we performed laparotomy (i.e., trauma induced) on rats and bled them to and maintained them at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with 3 times the volume of maximum bleedout with RL over 45 min followed by 2 times RL along with ATP-MgCl2 (50 mumol/kg body wt) over 95 min. Hepatocytes were isolated at 4, 17, and 27 h after resuscitation. P2-purinoceptor binding characteristics were determined by using [alpha-35S]ATP. Scatchard analysis revealed high-affinity and low-affinity receptor components in the hepatocytes isolated from sham-operated or hemorrhaged animals with or without ATP-MgCl2 infusion. ATP-MgCl2 ameliorated and subsequently restored the decreased maximum binding capacity (Bmax) of the high-affinity receptor component and significantly improved Bmax of the low-affinity receptor component. ATP-MgCl2 administration also produced a progressive enhancement in the affinity of the low-affinity receptor component. Thus the beneficial effects of ATP-MgCl2 observed after trauma-hemorrhage and resuscitation may be, in part, due to the restoration of P2-purinoceptor binding capacity and the enhancement of the receptor affinity.

Downregulation of hepatocyte P2-purinoceptor binding capacity after trauma-hemorrhage/resuscitation

1994 ◽  
Vol 266 (6) ◽  
pp. R1804-R1809
Author(s):  
M. S. Mahmoud ◽  
P. Wang ◽  
S. R. Hootman ◽  
S. S. Reich ◽  
I. H. Chaudry
Keyword(s):  
Dissociation Constants ◽  
Binding Capacity ◽  
Isolated Hepatocytes ◽  
Scatchard Analysis ◽  
Midline Laparotomy ◽  
Binding Characteristics ◽  
Shed Blood ◽  
P2 Purinoceptors ◽  
Altered Glucose ◽  
Affinity Receptor

Although P2-purinoceptors play an important role in the regulation of liver metabolism under normal conditions, it is not known if trauma-hemorrhage and resuscitation have any effects on such receptors. To study this, we performed a 5-cm midline laparotomy (i.e., trauma induced) on rats and then bled them to and maintained them at a mean arterial pressure of 40 mmHg until 40% of maximum bleedout volume was returned in the form of Ringer lactate (RL). The animals were then resuscitated with 3x the volume of shed blood with RL over 45 min followed by 2x RL over 95 min. Hepatocytes were isolated at the time of maximum bleedout or at 0, 4, 17, and 27 h after the completion of crystalloid resuscitation. P2-purinoceptor binding characteristics were determined in the isolated hepatocytes by using [alpha-35S]ATP. Scatchard analysis revealed high- and low-affinity components of P2-purinoceptors in hepatocytes from sham-operated as well as hemorrhaged and resuscitated animals. The maximum binding capacity (Bmax) of the high-affinity receptor component decreased at the time of maximum bleedout and at 4, 17, and 27 h after resuscitation. In addition to this, the Bmax of low-affinity receptor components also decreased at 4-27 h after resuscitation. In contrast, the dissociation constants of both receptor components were not altered. Because hemorrhagic shock produces abnormalities in glucose metabolism, the downregulation of hepatocyte P2-purinoceptor Bmax may be responsible for the altered glucose homeostasis under such conditions.

Burn-induced alterations in cardiac beta-adrenergic receptors

1992 ◽  
Vol 262 (5) ◽  
pp. H1585-H1591 ◽  
Author(s):  
T. M. Kaufman ◽  
J. W. Horton
Keyword(s):  
Guinea Pig ◽  
Guinea Pigs ◽  
Adrenergic Receptors ◽  
Burn Injury ◽  
Binding Capacity ◽  
Total Body Surface Area ◽  
Scatchard Analysis ◽  
Beta Adrenergic Receptors ◽  
Binding Studies ◽  
Beta Adrenergic

Previous studies in our laboratory have demonstrated that burn injury (45% total body surface area, 3rd-degree scald burn) diminishes contractile and relaxation function in the isolated perfused guinea pig heart. The mechanisms responsible for the burn-mediated dysfunction are not well understood. Therefore the purpose of this study was to examine the inotropic response to isoproterenol, a beta-adrenergic agonist, and burn-induced alterations in beta-adrenergic receptors (beta-AR) in adult guinea pig hearts. Isoproterenol dose-response curves were generated in isolated perfused hearts from sham-burned and burned guinea pigs. In addition, binding studies were performed using [125I]iodocyanopindolol on hearts from sham-burned and burned guinea pigs. Both the functional response and sensitivity to isoproterenol were significantly diminished 24 h after burn injury. beta-AR density (binding capacity, Bmax) and affinity were determined by Scatchard analysis. Agonist competition curves were performed in the presence or absence of 0.1 mM 5'-guanylyl imidodiphosphate. There was no difference in Bmax in membranes from sham-burned and burned hearts; however, the affinity of beta-AR was significantly decreased after burn injury compared with sham burn [dissociation constant = 32.5 +/- 1.9 (mean +/- SE), n = 10, vs. 26.7 +/- 1.7 pM, n = 10, P = 0.039]. Furthermore, the fraction of receptors in a high-affinity state (those functionally coupled to Gs protein) was significantly decreased after burn injury compared with sham burn (41.2 +/- 4.7%, n = 9, vs. 54 +/- 2%, n = 9, P = 0.023).(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotrophins: structural relatedness and receptor interactions

1991 ◽  
Vol 331 (1261) ◽  
pp. 255-258 ◽  
Keyword(s):  
Biological Response ◽  
Target Cells ◽  
Binding Studies ◽  
3 Dimensional ◽  
Receptor Complexes ◽  
High Affinity ◽  
Neurotrophin 3 ◽  
High Affinity Receptor ◽  
Affinity Receptor ◽  
Related Proteins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are structurally related proteins that allow the survival of specific populations of embryonic vertebrate neurons. The primary structure of these neurotrophins, deduced from their nucleotide sequences, indicates that all three are synthesized in the form of precursor proteins presumably allowing for appropriate folding, including the formation of disulphide bridges, cleavage and secretion. While no information is yet available on the 3-dimensional structures of the neurotrophins, results from binding studies using the three neurotrophins as ligands indicate that their receptors do recognize similarities, as well as differences, between them. High-affinity receptors, that presumably mediate the biological response, as well as low-affinity receptors are present on neurons responsive to the neurotrophins. Whereas a large excess of heterologous ligand is needed to reduce binding of a particular neurotrophin to its high affinity receptor, the same concentration of homologous or heterologous ligand similarly reduce the binding of any of the three neurotrophins to the low-affinity receptor. For all three, the low-affinity receptor appears to be the already characterized NGF low-affinity receptor that seems to be an integral part of the high-affinity receptor complexes. These results suggest that the regulation of neuronal survival by target cells can, in part, be explained by the release from these cells of limiting quantities of the structurally related neurotrophins, each being recognized by a specific high-affinity receptor complex located on the nerve terminals of the responsive neurons.

Available FSH receptors in adult rat testis in vivo

1990 ◽  
Vol 125 (2) ◽  
pp. 293-299 ◽  
Author(s):  
D. J. Yoon ◽  
D. Reggiardo ◽  
R. David
Keyword(s):  
Fsh Receptor ◽  
Binding Studies ◽  
Rat Testis ◽  
In Vivo Binding ◽  
Adult Rat ◽  
High Affinity ◽  
High Affinity Receptor ◽  
Intracardiac Injection ◽  
Affinity Receptor

ABSTRACT It has been suggested that the tight junctions formed between Sertoli cells during the peripubertal period constitute a barrier to circulating FSH in the adult testis, limiting its access to the lumen of the seminiferous tubules. There are also reports of FSH receptor-binding inhibitors. These obervations prompted us to study the extent of FSH receptor availability in vivo in the adult rat. Experimental rats were given an intracardiac injection of rat FSH (rFSH), and the occupied receptor was measured by radioimmunoassay of acid-released rFSH from the testis. In the saline-injected control animals, there were 247 fmol occupied FSH receptors/g testis, and as much as 1788 fmol of the unoccupied high-affinity receptors/g testis, as measured by in-vitro binding studies. After intracardiac injection of increasing amounts of rFSH (up to 606 pmol), receptor occupancy increased to a maximum plateau of only 448 fmol/g testis. In contrast, when rFSH was given by intratesticular injection in order to achieve pharmacological doses in the testis, the maximum binding was 662 fmol/g testis. Scatchard analysis of the in-vivo data revealed, however, that the maximum concentration of the high-affinity receptor was 452 fmol/g testis, a value concordant with the highest in-vivo binding observed in animals given intracardiac rFSH (448 fmol/g). A single injection of the hormone did not induce down-regulation of FSH receptors, regardless of the dose, whereas multiple injections of menotrophin were effective, at least to some extent. Despite the receptor loss, the immediate receptor availability was maintained, suggesting the presence of a receptor pool. In conclusion, in-vivo binding of FSH to its high-affinity receptor is limited in adult rat testis, and the available receptor concentration appears to be regulated so as to maintain a constant level. Journal of Endocrinology (1990) 125, 293–299

scholarly journals Different characteristics of solubilized lactogen receptors from livers of pregnant and non-pregnant female rats

1982 ◽  
Vol 203 (3) ◽  
pp. 653-662 ◽  
Author(s):  
Norio Sasaki ◽  
Yuko Tanaka ◽  
Yasuo Imai ◽  
Toshio Tsushima ◽  
Fukashi Matsuzaki
Keyword(s):  
Binding Capacity ◽  
Pregnant Female ◽  
Female Rats ◽  
Scatchard Analysis ◽  
Female Rat ◽  
Binding Studies ◽  
Pregnant Rats ◽  
Pregnant Rat ◽  
Stokes Radius ◽  
Rat Livers

Receptors specific for lactogenic hormones were solubilized by 1% (v/v) Triton X-100 from the crude particulate membrane fraction of livers of pregnant and non-pregnant female rats and the characteristics of both preparations were compared. Human 125I-labelled somatotropin was used for binding studies of lactogenic hormone. The solubilized receptor retained most of the characteristics noted in the particulate fraction. The binding of human 125I-labelled somatotropin to the solubilized receptor is a saturable process, depending on temperature and time. Scatchard analysis of displacement curves revealed similar affinity constants ranging from 1.02 × 109 to 1.20 × 109 1/mol, while the binding capacity was 4.5 times greater in the pregnant rat livers than in the non-pregnant female rat livers. The receptors for human 125I-labelled somatotropin from livers of non-pregnant and pregnant female rats were equally adsorbed onto a concanavalin-A-Sepharose column and were dissociated from the column with α-methyl-d-glucoside or α-methyl-d-mannoside in the same manner. By gel filtration on Sepharose 6B, however, the molecular sizes of the hepatic receptors were found to be different. The apparent Mr value was approx. 270000 with a Stokes‘ radius of 5.5nm in the non-pregnant female rats and approx. 330000 with a Stokes’ radius of 5.5nm in the pregnant rats. Furthermore, isoelectric-focusing experiments showed that a major part of the receptor from the non-pregnant female rat livers had a neutral pI (7.0—8.5), whereas that from pregnant-rat livers had an acidic pI (4.2—4.7). These data suggest that the increase in the lactogenic binding capacity in rat liver membranes during pregnancy may be associated with marked changes of the physicochemical properties of the receptors.

Expression of insulin receptors and of 60-kDa receptor substrate in rat mature and immature enterocytes

1997 ◽  
Vol 273 (1) ◽  
pp. G217-G226 ◽  
Author(s):  
J. P. Buts ◽  
N. De Keyser ◽  
S. Marandi ◽  
A. S. Maernoudt ◽  
E. M. Sokal ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Radioligand Binding ◽  
Binding Capacity ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Age Related ◽  
High Affinity Receptor

The mechanism(s) by which rat immature enterocytes exhibit increased responsiveness to insulin before weaning is unknown. Therefore, we have analyzed the distribution, ontogeny, and molecular properties of insulin receptors (IR) and of related substrates in immature and mature enterocytes. IR were studied by radioligand binding assays, cross-linking labeling, immunohistochemistry, and in vitro phosphorylated substrates by immunoprecipitation. Regardless of age, 125I-insulin binding to IR was five times higher in crypt cells than in villus cells and two times higher in the ileum than in the jejunum. Binding capacity to villus cells from sucklings (day 14) exceeded three times that of older animals (day 30 and day 60). Scatchard analysis of equilibrium binding data confirmed an age-related decrease in low- and high-affinity receptor classes without change in affinity constants. In concordance, both alpha- and beta-IR subunits were more abundant in immature than in mature membranes. In vitro, insulin elicited the phosphorylation of three membrane proteins (96, 60 and 42 kDa), whose signals were virtually inhibited by preincubating membranes with antireceptor monoclonal antibodies. By immunoprecipitation, the 60-kDa signal was rapidly detected as a tyrosine-phosphorylated protein, expressed in mature and immature membranes, and identified as a receptor substrate phosphorylated in vitro by the IR tyrosine kinase. In conclusion, 1) increased responsiveness of rat immature enterocytes to insulin could be related to high membrane concentrations of IR and 2) normal rat enterocytes express a 60-kDa phosphotyrosine protein identified as a direct substrate of the IR tyrosine kinase.

Cholecystokinin receptors and PANC-1 human pancreatic cancer cells

1993 ◽  
Vol 265 (1) ◽  
pp. G149-G155 ◽  
Author(s):  
J. P. Smith ◽  
C. A. Rickabaugh ◽  
P. J. McLaughlin ◽  
I. S. Zagon
Keyword(s):  
Pancreatic Cancer ◽  
Receptor Antagonist ◽  
Cancer Cells ◽  
Binding Capacity ◽  
Pancreatic Cancer Cell Line ◽  
Human Pancreatic Cancer ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
Cck Receptors ◽  
Pancreatic Cancer Cells

The gastrointestinal peptide cholecystokinin (CCK) has been shown to stimulate growth of human pancreatic adenocarcinoma in vitro and in vivo, although CCK receptors have not been identified in pancreatic cancer cells. The purpose of this study was to characterize the CCK receptors in pancreatic cancer cells and to correlate the receptor binding studies with the trophic action of CCK agonists and antagonists. With the use of homogenates of PANC-1 human pancreatic cancer cell line grown in culture, the binding of 125I-labeled CCK octapeptide (125I-CCK-8) was examined under various conditions to characterize the CCK receptor. Specific and saturable binding of 125I-CCK-8 was detected in PANC-1 cells; data were consistent with a single binding site. Scatchard analysis yielded a binding affinity [dissociation constant (Kd)] of 2.8 nM and a binding capacity of 26 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, the presence of protease inhibitors, and pH and was sensitive to Na+, K+, Mg2+, and ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Competition experiments indicated that L-365,260, a selective CCK-B (gastrin) receptor antagonist, was the most potent displacer of 125I-CCK-8, and no significant displacement of binding was found with the selective CCK-A receptor antagonist. Growth of PANC-1 cells in culture was stimulated by CCK at a concentration consistent with the Kd, and CCK-stimulated growth was inhibited by the CCK-B receptor antagonist (L-365,260) not the CCK-A receptor antagonist (L-364,718).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of the adenylyl cyclase activity in human endometrial membranes by VIP and related peptides

1993 ◽  
Vol 13 (2) ◽  
pp. 69-77 ◽  
Author(s):  
A. M. Bajo ◽  
L. G. Guijarro ◽  
M. G. Juarranz ◽  
P. Valenzuela ◽  
P. Martinez ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Binding Capacity ◽  
Maximal Activity ◽  
Human Endometrium ◽  
Cyclase Activity ◽  
Adenylyl Cyclase Activity ◽  
Maximal Stimulation ◽  
High Affinity Receptor ◽  
Affinity Receptor ◽  
Stimulation Of

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 μM GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0 ± 7.0 nM VIP, whereas the maximal activity (at 1 μM VIP)corresponded to an increase of about 140% with respect to basal values (7.5 ± 0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 μM) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED50 = 1.8 ± 1.4 nM) > VIP(ED50 = 25.0 ± 7.0 nM) > PHI (ED50 = 725.0 ± 127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of αs and αi subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of 125I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd=2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd = 0.43 μM, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium.

Factitious hyperthyroxinemia due to a monoclonal IgA in a case of multiple myeloma

1993 ◽  
Vol 39 (8) ◽  
pp. 1739-1742 ◽  
Author(s):  
K Cissewski ◽  
J D Faix ◽  
D Reinwein ◽  
A C Moses
Keyword(s):  
Multiple Myeloma ◽  
Binding Capacity ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Binding Studies ◽  
Free T4 ◽  
Total Thyroxine ◽  
Direct Binding ◽  
Hormone Binding Protein ◽  
Euthyroid Hyperthyroxinemia

Abstract A clinically euthyroid 53-year-old woman with an IgA-lambda-secreting multiple myeloma presented with increased serum concentrations of thyroid hormones. Laboratory studies revealed increased total thyroxine (T4) and triiodothyronine (T3) concentrations, a high-normal free T4 concentration, and a normal basal thyrotropin (TSH) concentration with a normal response to thyroliberin (TRH). Her serum concentration of IgA was 11,040 mg/L (normal range 900-4500 mg/L) and immunoelectrophoresis revealed it to be monoclonal. This monoclonal IgA bound both T4 and T3, as determined by serum immunoelectrophoresis and direct binding studies. Immunoelectrophoresis in the presence of [125I]T4 or [125I]T3 localized the radiolabeled iodothyronines to a band corresponding exactly to the precipitin arc of the monoclonal IgA. We performed direct binding studies with IgA purified by affinity chromatography with the lectin jacalin. Purified IgA (50 micrograms) bound both [125I]T4 (12.3%) and [125I]T3 (2.7%) specifically and in a dose-dependent manner. Scatchard analysis of competitive-binding data utilizing [125I]T4 and unlabeled T4 revealed a Kd of 2.2 x 10(-7) mol/L. The binding capacity for T4 was approximately 7 mumol/L. Thus, in this case of IgA-secreting myeloma, the monoclonal IgA acts as an additional thyroid hormone-binding protein in serum that interferes in the T4 and T3 RIAs. This is the first report of a monoclonal IgA producing an apparent euthyroid hyperthyroxinemia.

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