Synaptosomal T3 binding sites in rat brain: their localization on synaptic membrane and regional distribution

1983 ◽  
Vol 104 (2) ◽  
pp. 134-138 ◽  
Author(s):  
Yasuo Mashio ◽  
Mitsuo Inada ◽  
Kiyoshi Tanaka ◽  
Hitoshi Ishii ◽  
Koichi Naito ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Rat Brain ◽  
Binding Sites ◽  
Binding Capacity ◽  
Regional Distribution ◽  
Brain Regions ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Scatchard Analysis ◽  
Affinity Binding

Abstract. Previous studies have shown the existence of specific high affinity T3 binding sites in rat cerebrocortical synaptosomes. In this study, to define the localization of the binding sites, T3 binding to disrupted synaptic membrane fractions was compared with the binding to intact synaptosomes obtained from the rat cerebral cortex. Scatchard analysis revealed two orders of T3 binding sites with almost identical apparent dissociation constant (Kd) in synaptosomes and synaptic membrane fractions. However, the maximal binding capacity (MBC) of the higher affinity binding sites for synaptic membrane fractions was significantly greater than that for intact synaptosomes (4.2 ± 0.2 vs 3.0 ± 0.3 pg T3/mg protein). Regional distribution of the synaptosomal T3 binding sites in various areas of the rat brain was also studied. The Kd values were not significantly different in discrete brain regions. On the other hand, the MBCs of the higher affinity binding sites were greater in the cerebral cortex and hypothalamus (72.8 ± 1.4 and 64.8 ± 9.5 pg T3/g tissue) than in the cerebellum (22.1 ± 1.6 pg T3/g tissue). Similar difference was also observed in the lower affinity sites. These results indicate that the specific T3 binding sites in brain synaptosomes are localized mainly on synaptic membranes and that the MBC is different in discrete brain regions.

Hormone receptors in the epiphysial cartilage

1984 ◽  
Vol 103 (2) ◽  
pp. 125-131 ◽  
Author(s):  
K. W. Kan ◽  
R. L. Cruess ◽  
B. I. Posner ◽  
H. J. Guyda ◽  
S. Solomon
Keyword(s):  
Growth Plate ◽  
Binding Sites ◽  
Hormone Receptors ◽  
Binding Capacity ◽  
Specific Binding ◽  
Serum Insulin ◽  
Skeletal Growth ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Epiphysial Cartilage

ABSTRACT In order to assess which hormones may exert direct effects on skeletal growth at the epiphysial growth plate, the specific binding of hormones to the epiphysial cartilage of growing dogs and rabbits was studied. Membrane fractions obtained by centrifugation of homogenates prepared from dog and rabbit growth plate cartilage at 600, 15 000 and 105 000 g showed significant specific binding of serum insulin-like activity and insulin. Binding of growth hormone and prolactin by the three membrane fractions was negligible. Saturable binding sites for triiodothyronine could be demonstrated in nuclei from the dog growth plate. Nuclear binding showed an apparent Kd of 11 ±3·6 nmol/l and a maximum binding capacity of 4·1 ± 1·6 pmol/mg DNA, a level comparable to dog liver. Using a viable chondrocyte suspension prepared from dog epiphysial cartilage, specific steroid binding in the cells could be demonstrated for [3H]dexamethasone but not 17α-methyltrienolone, oestradiol-17β or 1α,25-di-hydroxycholecalciferol. Scatchard analysis of dexamethasone binding showed high affinity binding sites having a Kd of 1·2 ± 0·35 nmol/l and a capacity of 1700 sites/cell, and a low affinity binding with a Kd of 109 ± 57 nmol/l and a capacity of 24 000 sites/cell. Steroid competition for the specific binding showed the following sequence of affinity: dexamethasone > corticosterone > 11-deoxycortisol > testosterone > oestradiol-17β. Although all of the hormones examined except prolactin have well-established physiological effects on skeletal growth, our present results suggest that some of the hormonal effects observed in intact animals are secondary and do not involve receptor–hormone interaction in cartilage as such. J. Endocr. (1984) 103, 125–131

[125I]Iodomelatonin-binding sites in the pigeon brain: binding characteristics, regional distribution and diurnal variation

1991 ◽  
Vol 128 (3) ◽  
pp. 475-482 ◽  
Author(s):  
H. Yuan ◽  
S. F. Pang
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Regional Distribution ◽  
Mitochondrial Fraction ◽  
Brain Regions ◽  
Scatchard Analysis ◽  
Nuclear Fraction ◽  
Cytosol Fraction ◽  
Brain Membrane ◽  
Binding Characteristics

ABSTRACT The binding and pharmacological characteristics of melatonin-binding sites labelled by [125I]iodomelatonin in membrane preparations from the pigeon brain were determined. Specific binding of [125I]iodomelatonin in the membrane preparations of pigeon brain was rapid, stable, saturable and reversible. The [125I]iodomelatonin-binding sites had the following order of pharmacological affinities: melatonin > 6-chloromelatonin > N-acetylserotonin > > 5-hydroxytryptamine > tryptamine > 5 -methoxytryptophol, > 1 - acetylin dole-3-carboxytryptamine, 5-hydroxyindole-3-acetic acid, l-tryptophan and 3-acetylindole. Compounds known to act on serotonin receptors, adrenoceptors or cholinoceptors were inactive compared with melatonin. Of the various brain regions studied, melatonin binding was greatest in the hypothalamus, intermediate in the mid-brain, pons-medulla and telencephalon, and low in the cerebellum. Subcellular fraction studies indicated that 39% of the binding was located in the mitochondrial fraction, 34% in the nuclear fraction, 21% in the microsomal fraction and 5·6% in the cytosol fraction. Scatchard analysis of the membrane preparations revealed a dissociation constant (Kd) of 206·3±57·9 pmol/l and a total number of binding sites (Bmax) of 26·7±1·9 fmol/mg protein in the middle of the light period (mid-light). In addition, saturation studies demonstrated that [125I]iodomelatonin-binding sites in pigeon brain membrane preparations were 36·2% higher at mid-light (26·7±1·9 fmol/mg protein) than in the middle of the dark period (19·6±1·25 fmol/mg protein), with no significant variation in their binding affinities. Journal of Endocrinology (1991) 128, 475–482

scholarly journals [3H]-ouabain binding sites in rat brain: distribution and properties assessed by quantitative autoradiography.

1992 ◽  
Vol 40 (6) ◽  
pp. 771-779 ◽  
Author(s):  
A A Maki ◽  
D G Baskin ◽  
W L Stahl
Keyword(s):  
Nervous System ◽  
Rat Brain ◽  
Cardiac Glycoside ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Affinity Binding ◽  
Ouabain Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Kd Values

The anatomic distribution of high- and low-affinity cardiac glycoside binding sites in the nervous system is largely unknown. In the present study the regional distribution and properties of these sites were determined in rat brain by quantitative autoradiography (QAR). Two populations of cardiac glycoside binding sites were demonstrated with [3H]-ouabain, a specific inhibitor of Na,K-ATPases: (a) high-affinity binding sites with Kd values of 22-69 nM, which were blocked by erythrosin B, and (b) low-affinity binding sites with Kd values of 727-1482 nM. Sites with very low affinity for ouabain were not found by QAR. High- and low-affinity [3H]-ouabain binding sites were both found in all brain regions studied, including somatosensory cortex, thalamic and hypothalamic areas, medial forebrain bundle, amygdaloid nucleus, and caudate-putamen, although the distributions of high- and low-affinity sites were not congruent. Low-affinity [3H]-ouabain binding sites (Bmax = 222-358 fmol/mm2) were approximately twofold greater in number than high-affinity binding sites (Bmax = 76-138 fmol/mm2) in these regions of brain. Binding of [3H]-ouabain to both high- and low-affinity sites was blocked by Na+; however, low-affinity binding sites were less sensitive to inhibition by K+ (IC50 = 6.4 mM) than the high-affinity [3H]-ouabain binding sites (IC50 = 1.4 mM). The QAR method, utilizing [3H]-ouabain under standard conditions, is a valid method for studying modulation of Na,K-ATPase molecules in well-defined anatomic regions of the nervous system.

scholarly journals Platelet membrane alterations induced by the local anesthetic dibucaine

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 463-471 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Periodic Acid ◽  
Platelet Membrane ◽  
Actin Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Abstract Tertiary amine local anesthetics modify a variety of platelet membrane- related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high- affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)- stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.

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.

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