Quantitative Autoradiography as a Tool to Study Receptors in Neural Tissue. Studies on 3H-Ouabain Binding Sites and Correlation with Synaptic Protein Phosphorylation in Different Brain Areas

1985 ◽  
pp. 381-396 ◽  
Author(s):  
Fabio Benfenati ◽  
Luigi F. Agnati ◽  
Kjell Fuxe ◽  
Marlo Cimino ◽  
Nino Battistini ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Binding Sites ◽  
Neural Tissue ◽  
Quantitative Autoradiography ◽  
Synaptic Protein ◽  
Ouabain Binding ◽  
Brain Areas

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.

Quantitative autoradiography of [3H]ouabain binding sites in rat brain

1984 ◽  
Vol 322 (1) ◽  
pp. 189-193 ◽  
Author(s):  
Alexander C. Spyropoulos ◽  
Thomas C. Rainbow
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Quantitative Autoradiography ◽  
Ouabain Binding

[3H]SCH 23390 identifies D-1 binding sites in rat striatum and other brain areas

1986 ◽  
Vol 38 (12) ◽  
pp. 907-912 ◽  
Author(s):  
GAVIN J. KILPATRICK ◽  
PETER JENNER ◽  
C. DAVID MARSDEN
Keyword(s):  
Binding Sites ◽  
Sch 23390 ◽  
Rat Striatum ◽  
Brain Areas

Food intake alters muscle protein gain with little effect on Na(+)-K(+)-ATPase and myosin isoforms in suckled rats

1997 ◽  
Vol 272 (5) ◽  
pp. R1461-R1471 ◽  
Author(s):  
M. L. Fiorotto ◽  
T. A. Davis
Keyword(s):  
Food Intake ◽  
Binding Sites ◽  
Nutrient Intake ◽  
Muscle Protein ◽  
Fiber Type ◽  
Muscle Size ◽  
Myosin Isoforms ◽  
Ouabain Binding ◽  
Rat Pups ◽  
Dna Contents

Biochemical maturation accompanies the rapid accretion of skeletal muscle in early life. We wished to determine whether changes in muscle protein accretion, induced by variations in food intake, altered the biochemical maturation of the soleus and the extensor digitorum longus (EDL) muscles. Rat pups were suckled in litters of 4, 10, or 16 to induce differences in food intake. At 21 days of age, muscle protein and DNA were quantitated and biochemical maturation was assessed from measurement of [3H]ouabain-binding site abundance and myosin isoform composition. Differences in food intake produced a twofold range in body and muscle weights and protein and DNA contents. Protein accretion was more sensitive to nutrient intake in the soleus than in the EDL. Serum 3-5,3'-triiodothyronine (T3) and insulin concentrations decreased with a reduction in food intake. Total ouabain-binding sites were not altered in either muscle and were independent of muscle size. Differences in myosin isoform composition were more pronounced for the soleus than the EDL, but were relatively small in magnitude. These results demonstrate that, whereas postnatal muscle protein accretion and circulating hormone concentrations are sensitive to food intake, the biochemical maturation is resilient. The immature muscle does not exhibit the fiber type-specific responses to malnutrition typical of mature muscle.

scholarly journals The Na,K-ATPase-Dependent Src Kinase Signaling Changes with Mesenteric Artery Diameter

2018 ◽  
Vol 19 (9) ◽  
pp. 2489 ◽  
Author(s):  
Lin Zhang ◽  
Christian Aalkjaer ◽  
Vladimir Matchkov
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Isometric Force ◽  
Src Kinase ◽  
Ouabain Binding ◽  
Arterial Diameter ◽  
Small Arteries ◽  
Functional Changes ◽  
High Affinity ◽  
Different Diameters

Inhibition of the Na,K-ATPase by ouabain potentiates vascular tone and agonist-induced contraction. These effects of ouabain varies between different reports. In this study, we assessed whether the pro-contractile effect of ouabain changes with arterial diameter and the molecular mechanism behind it. Rat mesenteric small arteries of different diameters (150–350 µm) were studied for noradrenaline-induced changes of isometric force and intracellular Ca2+ in smooth muscle cells. These functional changes were correlated to total Src kinase and Src phosphorylation assessed immunohistochemically. High-affinity ouabain-binding sites were semi-quantified with fluorescent ouabain. We found that potentiation of noradrenaline-sensitivity by ouabain correlates positively with an increase in arterial diameter. This was not due to differences in intracellular Ca2+ responses but due to sensitization of smooth muscle cell contractile machinery to Ca2+. This was associated with ouabain-induced Src activation, which increases with increasing arterial diameter. Total Src expression was similar in arteries of different diameters but the density of high-affinity ouabain binding sites increased with increasing arterial diameters. We suggested that ouabain binding induces more Src kinase activity in mesenteric small arteries with larger diameter leading to enhanced sensitization of the contractile machinery to Ca2+.

Observations on ouabain binding and membrane phosphorylation by the sodium pump

1976 ◽  
Vol 193 (1112) ◽  
pp. 217-234 ◽  
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Kidney Cortex ◽  
Relative Molecular Mass ◽  
Type B ◽  
Ouabain Binding ◽  
Na Pump ◽  
Pump Mechanism ◽  
Morphological Differences ◽  
Equilibrium Dissociation

A study has been made of ouabain binding and the formation of phosphoprotein from ATP and inorganic phosphate (P i ) with plasma membranes from rabbit and guinea-pig kidney cortex. The aim of the work was first to see whether apparently conflicting results in the literature arise from membranes being prepared by different methods and, secondly, to evaluate the results in relation to the Na pump mechanism. Three different methods were used to prepare membranes, types A, Au and B. The preparations differed markedly when ouabain binding was supported by Mg alone both in the amount bound and in the affinity. Mgdependent binding was influenced by 1 mM P i but the extent of stimulation varied according to the preparations. The main effect of P i was to decrease the equilibrium dissociation constant marginally for type A membranes but eightfold for type B membranes. In contrast, the maximum number of binding sites was little affected. The membrane affinity for ouabain in relation to Mg and P i therefore depended on the method of preparation. In the reaction with Mg-ATP, type Au and B membranes were both phosphorylated to about the same extent. On the other hand, they reacted differently with P i , type B membranes being phosphorylated (in the presence of Mg and ouabain) to the same extent as with ATP, whereas under the same conditions, type Au membranes gave only 15 % of the phosphorylation found with ATP. The phosphoprotein, however formed, whether from ATP or P i , or type Au or type B membranes, migrated in the same way on gel electrophoresis to give a relative molecular mass of approximately 90000. With each preparation, over a tenfold range of ATPase activity, there was a constant value of 1.2 in the ratio of the maximum phosphorylation by ATP compared with the maximum number of ouabain-binding sites. These results show that membranes prepared in different ways exhibit some consistent properties of the Na pump but also striking anomalies. In view of likely morphological differences in the preparations, it is concluded that the inconsistent features, notably the responses to Mg and P i , are an unreliable guide to the pump mechanism.

scholarly journals Quantitative autoradiography of [3H]sulpiride binding sites in rat brain

1984 ◽  
Vol 51 (1) ◽  
pp. 47-53 ◽  
Author(s):  
Therese Ryan Jastrow ◽  
Eric Richfield ◽  
Margaret E. Gnegy
Keyword(s):  
Rat Brain ◽  
Binding Sites ◽  
Quantitative Autoradiography

Increased number of ouabain binding sites in lymphocytes from borderline hypertensives

1989 ◽  
Vol 19 (s1) ◽  
pp. 72-77 ◽  
Author(s):  
J. ROKKEDAL NIELSEN ◽  
K. E. PEDERSEN ◽  
N. A. KLITGAARD ◽  
T. JOHANSEN
Keyword(s):  
Binding Sites ◽  
Ouabain Binding
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