The rainbow trout skeletal muscle β-adrenergic system: characterization and signaling

2003 ◽  
Vol 284 (3) ◽  
pp. R689-R697 ◽  
Author(s):  
Michel B. Lortie ◽  
Thomas W. Moon
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Specific Binding ◽  
Adrenergic System ◽  
Camp Production ◽  
Binding Assays ◽  
Single Class ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Adrenergic Antagonist ◽  
Number Of Binding Sites

The presence and functionality of β-adrenoceptors (β-ARs) were examined in red (RM) and white muscle (WM) membranes isolated from the rainbow trout Oncorhynchus mykiss. Specific binding assays revealed the presence of a single class of binding sites with similar affinities in both muscle types ( K d in nM: 0.14 ± 0.03 and 0.18 ± 0.03 for RM and WM, respectively) but with a significantly higher number of binding sites in RM compared with WM (Bmax in fmol/mg protein: 3.22 ± 0.11 and 2.60 ± 0.13, respectively). Selective and nonselective β-adrenergic agonists (β-AAs) and antagonists indicated an atypical β-AR pharmacology. This result may represent a nonmammalian β-AR classification or, more likely, the presence of more than one β-AR subtype in trout muscles with similar affinities that could not be kinetically resolved. Adenylyl cyclase (ACase) assays showed a dose-dependent increase in cAMP production as concentrations of β2-AAs increased in both muscle membranes with significantly higher basal cAMP production in RM compared with WM (cAMP production in pmol cAMP · mg protein−1 · 10 min−1: 24.67 ± 3.06 and 9.64 ± 3.45, respectively). The agonist-induced increase in cAMP production was blocked by the β-adrenergic antagonist propranolol, while the ACase activator forskolin increased cAMP production by 7- to 14-fold above basal and ∼3-fold above all β-AAs tested. This study demonstrated the presence of atypical β2-ARs on RM and WM membranes of trout, suggesting that β2-AAs may be a tool to enhance protein accretion through this signaling pathway.

A putative cortisol receptor in the rainbow trout erythrocyte: stress prevents starvation-induced increases in specific binding of cortisol

1997 ◽  
Vol 200 (14) ◽  
pp. 2035-2043
Author(s):  
T Pottinger ◽  
I Brierley
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Negative Impact ◽  
Specific Binding ◽  
Nuclear Fraction ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Specific Binding Sites ◽  
Erythrocyte Binding ◽  
Number Of Binding Sites ◽  
Fold Excess

Binding sites for the steroid hormone cortisol, with characteristics typical of a steroid receptor, were detected in the rainbow trout (Oncorhynchus mykiss) erythrocyte. Binding of [3H]cortisol to a washed and purified erythrocyte suspension was saturable (Bmax=0.33±0.06 fmol per 2x10(6) cells; approximately 100±18 sites per cell; mean ± s.e.m., N=6), of high affinity (Kd=4.7±0.4 nmol l-1) and reversible in the presence of an excess of unlabelled ligand. Maximum levels of specific binding were observed within 60 min of the addition of [3H]cortisol at 4 °C and were stable for 2­3 h. Within 20 min of the addition of excess unlabelled ligand, 60 % of specifically bound [3H]cortisol had dissociated. Both dexamethasone and cortisol completely displaced specifically bound [3H]cortisol at 100-fold excess, whereas a 1000-fold excess of unlabelled cortisone, 11-ketotestosterone, oestradiol-17ß, testosterone and 17,20ß-dihydroxy-4-pregnen-3-one failed to displace specifically bound [3H]cortisol completely. Specific binding sites for [3H]cortisol were located predominantly (92 %) within the cytosolic fraction of the erythrocyte, with a trace amount of specific binding (8 %) detectable in the membrane fraction. No specific binding of [3H]cortisol was apparent in the erythrocyte nuclear fraction. A 7 day period of confinement stress resulted in no significant change in the number of erythrocyte cortisol-binding sites in rainbow trout, although plasma cortisol levels were significantly elevated in the stressed fish. However, in control unconfined fish, there was a progressive and significant increase in the amount of specifically bound cortisol per cell during the course of the experiment (from 0.097±0.030 to 0.260±0.070 fmol per 2x10(6) cells). A similar result was obtained when the experiment was repeated for confirmation. In both experiments, food was withheld from control and confined fish because of the negative impact of stress on appetite. The possibility that the increase in the number of erythrocyte cortisol-binding sites was related to the withdrawal of food was tested by quantifying the amount of specifically bound cortisol in erythrocytes over a 14 day period in unstressed rainbow trout maintained on normal rations and in unstressed fish from which food was withheld. A significant increase in the amount of specifically bound cortisol was observed with time in the fasted fish (from 0.33±0.07 to 0.53±0.03 fmol per 2x10(6) cells). These data suggest that the abundance of erythrocyte cortisol-binding sites in trout is a function of nutritional status and that stress opposes a fasting-induced increase in the number of binding sites.

Endothelin ETA and ETB receptors in postnatal intestine

2001 ◽  
Vol 280 (4) ◽  
pp. G555-G562 ◽  
Author(s):  
Craig A. Nankervis ◽  
David J. Dunaway ◽  
Charles E. Miller
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Ischemia Reperfusion ◽  
Age Groups ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Site Model ◽  
Number Of Binding Sites ◽  
Competitive Binding Assays

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with 125I-labeled ET-1 in membranes from endothelium-denuded (E−) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive125I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E+) and E− vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ETAreceptor antagonist BQ-123, competitive 125I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E+ vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ETB receptor.

Specific [3H]spiperone binding sites in the pituitary of rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus)

1995 ◽  
Vol 73 (5) ◽  
pp. 585-593 ◽  
Author(s):  
Robert J. Omeljaniuk
Keyword(s):  
Rainbow Trout ◽  
Specific Binding ◽  
Developmental Differences ◽  
Pituitary Hormone ◽  
Pars Distalis ◽  
Single Class ◽  
Neurointermediate Lobe ◽  
Goldfish Carassius Auratus ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Nonspecific Effects

Dopamine, a catecholamine neurohormone, modulates pituitary hormone release in teleost fishes and other vertebrates. The existence and binding parameters of a pituitary dopamine–neuroleptic receptor from trout were examined and compared with those from goldfish. Pituitary homogenate was incubated with [3H]spiperone (D2 antagonist) under several experimental paradigms; incubations were terminated by filtration and bound 3H radioactivity was assessed by liquid scintillation spectroscopy. Specific binding of [3H]spiperone was tissue dependent. Equilibrium displacement analyses using domperidone (D2 antagonist) indicated a single class of binding site (LIGAND) with Kd = 2.49 ± 0.89 μM and a capacity of 3.10 ± 0.45 nmol/mg protein; the goldfish Kd and capacity were both significantly (p < 0.05) larger: Kd = 4.63 ± 0.30 μM and capacity = 20.66 ± 2.03 nmol/mg protein. The Kd and capacity for the trout pars distalis (2.45 ± 0.33 μM and 3.27 ± 0.24 nmol/mg protein, respectively) did not differ significantly (p < 0.05) from that of the neurointermediate lobe (2.50 ± 0.08 μM and 3.58 ± 0.56 nmol/mg protein, respectively). Dopamine D2 receptor ligands differentially displaced [3H]spiperone from the trout pituitary, while D1 ligands, a D4 ligand, and a 5-hydroxytryptamine (5HT2) receptor antagonist had only small nonspecific effects. Comparison of the trout and goldfish pituitary dopamine–neuroleptic receptor indicates conservation of receptor affinity (Kd); however, differences in receptor numbers and in the distribution of receptors between the pars distalis and neurointermediate lobe in the two species may be due in part to species or developmental differences, and may reflect differences in the role(s) and degrees of influence of dopamine in these fishes.Key words: pituitary, dopamine, receptor, rainbow trout, goldfish.

scholarly journals Leukemic B-cell precursors constitutively express functional receptors for human interleukin-1

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 761-776 ◽  
Author(s):  
FM Uckun ◽  
DE Myers ◽  
AS Fauci ◽  
M Chandan-Langlie ◽  
JL Ambrus
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Binding Sites ◽  
Specific Binding ◽  
Lymphoblastic Leukemia ◽  
Interleukin 1 ◽  
Receptor Occupancy ◽  
Binding Assays ◽  
Single Class ◽  
B Cell Precursors

Abstract This study analyzes the expression of functional interleukin-1 (IL-1) receptors on leukemic B-cell precursors (BCPs) from B-cell precursor acute lymphoblastic leukemia (BCP ALL) patients. We first investigated the specific binding of 125I-labeled recombinant IL-1 (125I-rIL-1) (4 x 10(17) cpm/mol) to fresh marrow blasts from 11 BCP ALL patients. In five of 11 cases, the binding of 125I-rIL-1 was significantly blocked by excess cold rIL-1. In these five cases, the cell-bound radioactivity ranged from 146 cpm/10(6) cells to 2,412 cpm/10(6) cells (mean +/- SE = 782 +/- 414 cpm/10(6) cells), indicating that 4 to 60 femtomols (mean +/- SE = 20 +/- 10 femtomols) of 125I-rIL-1 specifically bound per 10(7) cells. The estimated number of 125I-rIL-1 molecules bound per cell ranged from 219 to 3,618 (mean +/- SE = 1173 +/- 621). In all five cases, BCP colony formation was stimulated by 10 ng/mL (570 femtomolar) rIL-1, and the background-subtracted colony numbers ranged from 130 to 298 (mean +/- SE = 226 +/- 31). In contrast, no stimulation was observed in six cases that showed no significant 125I-rIL-1 binding. Hence, there was a high correlation between 125I-rIL-1 binding and IL-1 responsiveness, indicating that functional IL-1 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for leukemic BCPs from two IL-1-responsive BCP ALL cases yielded straight linear regression lines, indicating the existence of a single class of 132 to 154 high affinity IL-1 receptors/cell. The apparent affinity constants (Ka) values ranged from 5.2 x 10(9) mol/L-1 to 1.2 x 10(10) mol/L-1. Notably, the concentrations of IL-1 required for half-maximal receptor occupancy (kd = 83 pmol/L to 190 pmol/L) were approximately three orders of magnitude higher than those needed to elicit a half-maximal proliferative response of leukemic BCPs in colony assays (0.1 to 1.0 ng/mL = 5.7 to 57 femtomolar), indicating that only a small fraction of IL-1 receptors need to be occupied to stimulate leukemic BCPs. Notably, IL-1 unresponsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines (REH, KM-3) did not exhibit any significant IL-1 binding (less than 10 IL-1 binding sites/cell), and two additional IL-1 unresponsive BCP ALL cell lines (NALM-6, HPB-NULL) expressed only 24 to 54 IL-1 binding sites/cell with a Ka of 7.8 to 9.8 x 10(9) mol/L- 1.

Characteristics of atrial natriuretic hormone receptors in human pheochromocytomas

1990 ◽  
Vol 122 (6) ◽  
pp. 740-744 ◽  
Author(s):  
J. Eurin ◽  
A. Carayon ◽  
M. A. Zongazo ◽  
F. Masson ◽  
C. Barthelemy ◽  
...  
Keyword(s):  
Binding Sites ◽  
Hormone Receptors ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Single Class ◽  
Natriuretic Hormone ◽  
Number Of Binding Sites ◽  
Normal Human ◽  
Atrial Natriuretic Hormone ◽  
Atrial Natriuretic

Abstract. The presence of functional receptors for human atrial natriuretic hormone in human pheochromocytomas was recently reported. The present study reports the binding of hANH as measured by Scatchard analysis in 4 human adrenal glands and in 5 human pheochromocytomas. Binding assays using [3H]ANH revealed a single class of high-affinity binding sites for hANH in both tissues. Human pheochromocytomas present a lower number of binding sites than normal human adrenal gland (Bmax of 7.1±2.1 vs 33.6±6.9 fmol/mg protein, respectively). However, the decreased number of ANH receptors was not paralleled by modifications of tissular cyclic GMP (cGMP). Moreover, plasma hANH concentrations in 7 patients with pheochromocytomas (20.2±2.7 pmol/l) were statistically higher than those obtained in 25 normal control humans (8.1±0.6 pmol/l, p<0.001). We also demonstrated the presence of immunoreactive ANH in the tumour itself.

Increase in number of myocardial [3H]BAY K 8644 binding sites during adult maturation of rat

1990 ◽  
Vol 68 (7) ◽  
pp. 877-881 ◽  
Author(s):  
Srisala Navaratnam ◽  
Jagdish C. Khatter
Keyword(s):  
Calcium Channels ◽  
Binding Sites ◽  
Specific Binding ◽  
Bay K 8644 ◽  
Sprague Dawley ◽  
Single Class ◽  
Voltage Gated ◽  
Sarcolemmal Membrane ◽  
Voltage Gated Calcium Channels ◽  
Number Of Binding Sites

In the present study we investigated the binding properties of [3H]BAY K 8644 to the purified sarcolemmal membrane, isolated from 2- and 12-month old Sprague–Dawley rats. Specific binding of [3H]BAY K 8644 was saturable and the Scatchard plot analysis revealed a single class of binding sites in purified sarcolemmal membrane. The estimated maximum number of binding sites in the membrane of 12-month-old rat was 2.4 ± 0.1 pmol/mg protein, which was significantly greater than the maximum number of binding sites in 2-month-old rats (1.7 ± 0.2 pmol/mg protein). The affinity to bind [3H]BAY K 8644 was, however, reduced in older rats (KD, 14.5 ± 0.8 vs. 4.8 ± 0.3 nM). Measurement of activities of sarcolemmal and subcellular marker enzymes showed that the purification of membrane was virtually identical in two age groups. This would suggest that membrane purity was not a contributing factor to the observed increase in [3H]BAY K 8644 receptor density. Since dihydropyridine receptor sites are very likely to represent voltage-gated calcium channels of sarcolemma, it is concluded that the density of myocardial voltage-gated calcium channels increases during adult maturation.Key words: BAY K 8644, Ca2+ channel, maturation, age.

Growth hormone receptors in the liver and osmoregulatory organs of rainbow trout: characterization and dynamics during adaptation to seawater

1991 ◽  
Vol 130 (3) ◽  
pp. 425-433 ◽  
Author(s):  
T. Sakamoto ◽  
T. Hirano
Keyword(s):  
Growth Hormone ◽  
Rainbow Trout ◽  
Binding Sites ◽  
Chum Salmon ◽  
Hormone Receptors ◽  
Sea Water ◽  
Specific Binding ◽  
Head Kidney ◽  
Scatchard Analysis ◽  
Single Class

ABSTRACT Specific binding sites for chum salmon growth hormone (sGH) were identified in the membranes obtained from tissues of rainbow trout. Specific binding of 125I-labelled sGH (% per mg protein) was found in the liver (37%), ovary (6%), brain (6%), gill (4%), intestine (4%) and posterior body kidney (4%). Specific binding was not significant in head kidney, anterior body kidney, spleen, heart, skeletal muscle or skin. Scatchard analyses demonstrated the presence of a single class of high-affinity low-capacity receptors in the liver, gill, intestine and kidney. The association constants for the membranes from liver, gill, intestine and kidney were of the same order (1 litre/nmol). Chum salmon prolactin did not inhibit the binding of 125I-labelled sGH to receptors in the liver, gill, intestine and kidney. Transfer of rainbow trout from fresh water to 80% seawater evoked a rise in plasma concentration of GH and a significant decrease in the GH binding to the liver membranes after 1 day. Binding in the gill and kidney was not altered significantly. Membranes were treated with 4 mol MgCl2/l to remove bound GH from the receptors, and the results indicated that the reduction in binding in the liver after transfer to sea-water was probably due to receptor occupancy by increased endogenous GH. The occupancy of liver GH-binding sites was maximal 4 days after transfer. Total (MgCl2-treated) binding sites in the liver increased significantly 14 days after transfer. Scatchard analysis indicated that receptors were altered in capacity without changes in binding affinity. Although GH may also directly affect osmoregulatory organs through their GH receptors, the present results indicate the likelihood of at least partial mediation by the liver of the seawater-adapting action of GH in the rainbow trout. Journal of Endocrinology (1991) 130, 425–433

scholarly journals Leukemic B-cell precursors constitutively express functional receptors for human interleukin-1

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 761-776
Author(s):  
FM Uckun ◽  
DE Myers ◽  
AS Fauci ◽  
M Chandan-Langlie ◽  
JL Ambrus
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Binding Sites ◽  
Specific Binding ◽  
Lymphoblastic Leukemia ◽  
Interleukin 1 ◽  
Receptor Occupancy ◽  
Binding Assays ◽  
Single Class ◽  
B Cell Precursors

This study analyzes the expression of functional interleukin-1 (IL-1) receptors on leukemic B-cell precursors (BCPs) from B-cell precursor acute lymphoblastic leukemia (BCP ALL) patients. We first investigated the specific binding of 125I-labeled recombinant IL-1 (125I-rIL-1) (4 x 10(17) cpm/mol) to fresh marrow blasts from 11 BCP ALL patients. In five of 11 cases, the binding of 125I-rIL-1 was significantly blocked by excess cold rIL-1. In these five cases, the cell-bound radioactivity ranged from 146 cpm/10(6) cells to 2,412 cpm/10(6) cells (mean +/- SE = 782 +/- 414 cpm/10(6) cells), indicating that 4 to 60 femtomols (mean +/- SE = 20 +/- 10 femtomols) of 125I-rIL-1 specifically bound per 10(7) cells. The estimated number of 125I-rIL-1 molecules bound per cell ranged from 219 to 3,618 (mean +/- SE = 1173 +/- 621). In all five cases, BCP colony formation was stimulated by 10 ng/mL (570 femtomolar) rIL-1, and the background-subtracted colony numbers ranged from 130 to 298 (mean +/- SE = 226 +/- 31). In contrast, no stimulation was observed in six cases that showed no significant 125I-rIL-1 binding. Hence, there was a high correlation between 125I-rIL-1 binding and IL-1 responsiveness, indicating that functional IL-1 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for leukemic BCPs from two IL-1-responsive BCP ALL cases yielded straight linear regression lines, indicating the existence of a single class of 132 to 154 high affinity IL-1 receptors/cell. The apparent affinity constants (Ka) values ranged from 5.2 x 10(9) mol/L-1 to 1.2 x 10(10) mol/L-1. Notably, the concentrations of IL-1 required for half-maximal receptor occupancy (kd = 83 pmol/L to 190 pmol/L) were approximately three orders of magnitude higher than those needed to elicit a half-maximal proliferative response of leukemic BCPs in colony assays (0.1 to 1.0 ng/mL = 5.7 to 57 femtomolar), indicating that only a small fraction of IL-1 receptors need to be occupied to stimulate leukemic BCPs. Notably, IL-1 unresponsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines (REH, KM-3) did not exhibit any significant IL-1 binding (less than 10 IL-1 binding sites/cell), and two additional IL-1 unresponsive BCP ALL cell lines (NALM-6, HPB-NULL) expressed only 24 to 54 IL-1 binding sites/cell with a Ka of 7.8 to 9.8 x 10(9) mol/L- 1.

Fasting alters somatostatin binding to liver membranes of rainbow trout (Oncorhynchus mykiss)

1996 ◽  
Vol 150 (2) ◽  
pp. 179-186 ◽  
Author(s):  
M J Pesek ◽  
M A Sheridan
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
High Affinity ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
C Terminus ◽  
Liver Membranes

Abstract Somatostatins are a diverse family of peptides that influence various aspects of animal growth, development, and metabolism. Recent work in our laboratory has shown that somatostatins stimulate hepatic lipolysis in rainbow trout. In this study we characterized somatostatin-binding sites in trout hepatic membrane preparations. We also examined changes in binding characteristics brought about by food deprivation. Binding of [Tyr11]-somatostatin-14 (SS-14) was saturable, reversible, and time- and temperature-dependent. Under optimal conditions, [Tyr11]-SS-14 specific binding averaged 5·7 ± 0·3%. While SS-14 and SS-28 (an N-terminally extended form of SS-14 and derived from the same gene as SS-14) displaced [Tyr11]-SS-14 specific binding (ED50 values of approximately 50 nm and 100 nm respectively), salmon SS-25 (containing [Tyr7,Gly10]-SS-14 at its C terminus and presumably derived from a gene different from that giving rise to SS-14/SS-28), except at pharmacological concentrations, did not. Significant specific binding was also detected in brain, esophagus, stomach, upper and lower intestine, pancreas, and adipose tissue. Scatchard analysis suggested the existence of two classes of hepatic somatostatin-binding sites: a high-affinity site with a Kd of 23 nm and Bmax of 1·4 pmol/mg protein and a low-affinity site with a Kd of 379 nm and Bmax of 4·9 pmol/mg protein. Fasting resulted in reduced growth and elevated plasma levels of SS-14 compared with fed animals. SS-14 binding capacity of the high-affinity class in liver membranes isolated from fasted fish increased by 120% over that from fed counter-parts. No difference in Kd for the high-affinity binding class or in either Kd or Bmax of the low-affinity class was noted between fasted and fed animals. These data support the role of the liver as a target of somatostatin and suggest that fasting enhances hepatic sensitivity to SS-14 binding. Journal of Endocrinology (1996) 150, 179–186

Sign in / Sign up

Export Citation Format

Share Document