scholarly journals Ouabain binding and coupled sodium, potassium, and chloride transport in isolated transverse tubules of skeletal muscle.

1979 ◽  
Vol 74 (3) ◽  
pp. 335-349 ◽  
Author(s):  
Y H Lau ◽  
A H Caswell ◽  
M Garcia ◽  
L Letellier
Keyword(s):  
Binding Sites ◽  
Chloride Transport ◽  
Slow Phase ◽  
Ouabain Binding ◽  
Fast Phase ◽  
Highly Active ◽  
Tubular Lumen ◽  
Number Of Binding Sites ◽  
T Tubules ◽  
Low K

The affinity and number of binding sites of [3H]ouabain to isolated transverse (T) tubules were determined in the absence and presence of deoxycholate. In both conditions the KD was approximately 53 nM while deoxycholate increased the number of binding sites from 3.5 to 37 pmol/mg protein. We concluded that the ouabain binding sites were located primarily on the inside of the isolated vesicle and that the vesicles were impermeable to ouabain. ATP induced a highly active Na+ accumulation by the T tubules which increased Na+ in the T tubular lumen by almost 200 nmol/mg protein. The accumulation had an initial fast phase lasting 2-3 min and a subsequent slow phase which continued for at least 40 min. The rate of the initial fast phase indicated a turnover number of 20 Na+/s. The Na+ accumulation was prevented by monensin but was unaffected by valinomycin. Ouabain did not influence Na+ uptake, but digitoxin inhibited it. At low K+ the accumulation of Na+ was reduced 3.7-fold below the value at 50 mM K+. 86Rb, employed as a tracer to detect K+, showed a first phase of K+ release while Na+ was accumulated. After 2-3 min, K+ was reaccumulated while Na+ continued to increase in the lumen. T tubules accumulated Cl- on addition of ATP. This suggested that ATP initiated an exchange of Na+ for K+ followed by uptake of Na+ and K+ accompanied by Cl-.

scholarly journals Two Temporal Phases of Light Adaptation in Retinal Rods

2002 ◽  
Vol 119 (2) ◽  
pp. 129-146 ◽  
Author(s):  
Peter D. Calvert ◽  
Victor I. Govardovskii ◽  
Vadim Y. Arshavsky ◽  
Clint L. Makino
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Light Adaptation ◽  
Dynamic Range ◽  
Continuous Illumination ◽  
Slow Phase ◽  
Fast Phase ◽  
Slow Adaptation ◽  
Adaptation Phase

Vertebrate rod photoreceptors adjust their sensitivity as they adapt during exposure to steady light. Light adaptation prevents the rod from saturating and significantly extends its dynamic range. We examined the time course of the onset of light adaptation in bullfrog rods and compared it with the projected onset of feedback reactions thought to underlie light adaptation on the molecular level. We found that adaptation developed in two distinct temporal phases: (1) a fast phase that operated within seconds after the onset of illumination, which is consistent with most previous reports of a 1–2-s time constant for the onset of adaptation; and (2) a slow phase that engaged over tens of seconds of continuous illumination. The fast phase desensitized the rods as much as 80-fold, and was observed at every light intensity tested. The slow phase was observed only at light intensities that suppressed more than half of the dark current. It provided an additional sensitivity loss of up to 40-fold before the rod saturated. Thus, rods achieved a total degree of adaptation of ∼3,000-fold. Although the fast adaptation is likely to originate from the well characterized Ca2+-dependent feedback mechanisms regulating the activities of several phototransduction cascade components, the molecular mechanism underlying slow adaptation is unclear. We tested the hypothesis that the slow adaptation phase is mediated by cGMP dissociation from noncatalytic binding sites on the cGMP phosphodiesterase, which has been shown to reduce the lifetime of activated phosphodiesterase in vitro. Although cGMP dissociated from the noncatalytic binding sites in intact rods with kinetics approximating that for the slow adaptation phase, this hypothesis was ruled out because the intensity of light required for cGMP dissociation far exceeded that required to evoke the slow phase. Other possible mechanisms are discussed.

Effect of methacholine on Na+ pump activity and ion content of dispersed avian salt gland cells

1981 ◽  
Vol 241 (1) ◽  
pp. R77-R86
Author(s):  
S. R. Hootman ◽  
S. A. Ernst
Keyword(s):  
Binding Sites ◽  
Gland Cell ◽  
Salt Gland ◽  
Ion Content ◽  
Ouabain Binding ◽  
Gland Cells ◽  
Na Pump ◽  
Pump Activity ◽  
Number Of Binding Sites ◽  
Dissociated Cells

The effects of the cholinergic agonist methacholine chloride (MCh) on cellular ion content and Na+ pump activity of dissociated duck salt gland cells were studied. Dispersed salt gland cells regulate intracellular ion levels in a ouabain-sensitive manner. MCh (0.5 mM) caused no detectable change in cell Na+ levels over the first 10 min of exposure of cells to the agonist but elicited decreases of 23 and 13%, respectively, in intracellular Cl- and K+ content. The rate of turnover of salt gland cell plasmalemmal Na+ pumps, as measured by [3H]ouabain binding to the dissociated cells, was markedly stimulated by 0.5 mM MCh, although the total number of binding sites at equilibrium remained unchanged. Replacement of medium Na+ with choline abolished the MCh-stimulated increase in ouabain binding but had no effect on the rate of glycoside binding in the absence of the agonist. Substitution of Cl- in the medium by NO3-, SO42-, or benzene sulfonate- reduced the stimulated component of Na+ pump turnover by 85-90%. Addition of 1 mM furosemide to the medium abolished the increase in ouabain binding and ouabain-sensitive oxygen consumption observed after exposure of salt gland cells to MCh. These data are consistent with the hypothesis that cholinergic stimulation of salt gland cells triggers a Cl--dependent uptake of Na+, which elicits a compensatory increase in Na+ pump turnover. In addition, the decrease in cellular Cl- content caused by MCh suggests that the agonist either directly or indirectly mediates an efflux of Cl- from the cells.

Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species

2003 ◽  
Vol 285 (2) ◽  
pp. C319-C326 ◽  
Author(s):  
Xiaoming Zhou ◽  
Wu Yin ◽  
Sonia Q. Doi ◽  
Shawn W. Robinson ◽  
Kunio Takeyasu ◽  
...  
Keyword(s):  
Binding Sites ◽  
Regulatory Element ◽  
Protein Abundance ◽  
Oxygen Species ◽  
Ouabain Binding ◽  
Direct Stimulation ◽  
Negative Regulatory Element ◽  
Low K ◽  
Cat Expression ◽  
Stimulation Of

The signaling pathway that transduces the stimulatory effect of low K+ on the biosynthesis of Na,K-ATPase remains largely unknown. The present study was undertaken to examine whether reactive oxygen species (ROS) mediated the effect of low K+ in Madin-Darby canine kidney (MDCK) cells. Low K+ increased ROS activity in a time- and dose-dependent manner, and this effect was abrogated by catalase and N-acetylcysteine (NAC). To determine the role of ROS in low-K+-induced gene expression, the cells were first stably transfected with expression constructs in which the reporter gene chloramphenicol acetyl transferase (CAT) was under the control of the avian Na,K-ATPase α-subunit 1.9 kb and 900-bp 5′-flanking regions that have a negative regulatory element. Low K+ increased the CAT expression in both constructs. Catalase or NAC inhibited the effect of low K+. To determine whether the increased CAT activity was mediated through releasing the repressive effect or a direct stimulation of the promoter, the cells were transfected with a CAT expression construct directed by a 96-bp promoter fragment that has no negative regulatory element. Low K+ also augmented the CAT activity expressed by this construct. More importantly, both catalase and NAC abolished the effect of low K+. Moreover, catalase and NAC also inhibited low-K+-induced increases in the Na,K-ATPase α1- and β1-subunit protein abundance and ouabain binding sites. The antioxidants had no significant effect on the basal levels of CAT activity, protein abundance, or ouabain binding sites. In conclusion, low K+ enhances the Na,K-ATPase gene expression by a direct stimulation of the promoter activity, and ROS mediate this stimulation and also low-K+-induced increases in the Na,K-ATPase protein contents and cell surface molecules.

The Estimation of the Number of Binding Sites for Antibody on Antigen Molecules with Reference to Factor VIII and its Antibody

1976 ◽  
Vol 35 (02) ◽  
pp. 274-288 ◽  
Author(s):  
Judith Pool ◽  
Rosemary Biggs ◽  
R. G Miller
Keyword(s):  
Factor Viii ◽  
Binding Sites ◽  
Theoretical Basis ◽  
Human Antibody ◽  
Rabbit Antibody ◽  
Number Of Binding Sites ◽  
Theoretical Considerations

SummaryThe theoretical basis for determining the number of antibody sites on antigen molecules is examined. The theoretical considerations are applied to factor VIII molecules. Examples based on data available at the Oxford Haemophilia Centre are calculated to illustrate the approach. It is concluded that there are few sites on each factor VIII molecule for human antibody. The three antibodies for which reasonable data were available suggest 1–3 sites for human antibody. The data for rabbit antibody suggest 5–6 sites per factor VIII molecule.

Abnormal Platelet Serotonin Uptake and Binding Sites in Myeloproliferative Disorders

1984 ◽  
Vol 51 (03) ◽  
pp. 349-353 ◽  
Author(s):  
C Caranobe ◽  
P Sié ◽  
F Fernandez ◽  
J Pris ◽  
S Moatti ◽  
...  
Keyword(s):  
Binding Sites ◽  
High Capacity ◽  
Specific Inhibitor ◽  
Myeloproliferative Disorders ◽  
Normal Subjects ◽  
Binding Data ◽  
Full Explanation ◽  
Number Of Binding Sites ◽  
5 Ht Uptake ◽  
Kinetics Of

SummaryA simultaneous investigation of the kinetics of serotonin (5 HT) uptake and of binding sites was carried out in the platelets of normal subjects and of 10 patients affected with various types of myeloproliferative disorders (MD). The 5 HT uptake was analysed according to the Lineweaver-Burk and the Eadie-Hofstee methods. With the two methods, the patient’s platelets exhibited a dramatic reduction of the Vi max and of the Km; in some patients the Eadie-Hofstee analysis revealed that a passive diffusion phenomenon is superimposed on the active 5 HT uptake at least for the higher concentration used. The binding data were analysed with the Scatchard method. Two classes of binding sites (high affinity - low capacity, low affinity - high capacity) were found in normal subjects and patients. Pharmacological studies with imipramine, a specific inhibitor of 5 HT uptake, suggested that both the sites are involved in 5 HT uptake. The number of both binding sites was significantly decreased in patient’s platelets while the affinity constants of these binding sites were not significantly reduced in comparison with those of the control subjects. No correlations were found between Vi max, Km and the number of binding sites. These results suggest that a reduction in the number of platelet membrane acceptors for 5 HT commonly occurs in myeloproliferative disorders but does not provide a full explanation of the uptake defect.

Possible cues driving context-specific adaptation of optocollic reflex in pigeons (Columba livia)

2012 ◽  
Vol 107 (2) ◽  
pp. 704-717 ◽  
Author(s):  
Henri Gioanni ◽  
Pierre-Paul Vidal
Keyword(s):  
Columba Livia ◽  
Motor Command ◽  
Slow Phase ◽  
Gaze Stabilization ◽  
Specific Adaptation ◽  
Body Vibration ◽  
Fast Phase ◽  
Context Specific ◽  
Context Cues

Context-specific adaptation (Shelhamer M, Clendaniel R. Neurosci Lett 332: 200–204, 2002) explains that reflexive responses can be maintained with different “calibrations” for different situations (contexts). Which context cues are crucial and how they combine to evoke context-specific adaptation is not fully understood. Gaze stabilization in birds is a nice model with which to tackle that question. Previous data showed that when pigeons ( Columba livia) were hung in a harness and subjected to a frontal airstream provoking a flying posture (“flying condition”), the working range of the optokinetic head response [optocollic reflex (OCR)] extended toward higher velocities compared with the “resting condition.” The present study was aimed at identifying which context cues are instrumental in recalibrating the OCR. We investigated that question by using vibrating stimuli delivered during the OCR provoked by rotating the visual surroundings at different velocities. The OCR gain increase and the boost of the fast phase velocity observed during the “flying condition” were mimicked by body vibration. On the other hand, the newly emerged relationship between the fast-phase and slow-phase velocities in the “flying condition” was mimicked by head vibration. Spinal cord lesion at the lumbosacral level decreased the effects of body vibration, whereas lesions of the lumbosacral apparatus had no effect. Our data suggest a major role of muscular proprioception in the context-specific adaptation of the stabilizing behavior, while the vestibular system could contribute to the context-specific adaptation of the orienting behavior. Participation of an efferent copy of the motor command driving the flight cannot be excluded.

Effects of cations on binding of human choriogonadotropin

1988 ◽  
Vol 66 (12) ◽  
pp. 1258-1264
Author(s):  
Patrick J. McIlroy
Keyword(s):  
Binding Sites ◽  
Regulatory Protein ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Human Choriogonadotropin ◽  
Receptor Sites ◽  
Number Of Binding Sites ◽  
Receptor Number ◽  
Guanine Nucleotide Regulatory Protein ◽  
Low Ionic Strength

The effect of various salts on the binding of human choriogonadotropin to rat luteal membranes has been examined. Increasing salt concentrations had biphasic effects, initially increasing binding, then decreasing it. With NaCl, these effects were on both the affinity and the number of receptor sites. The affinity increased with increasing NaCl concentrations, to a maximum at 40 mM, and then decreased. Above 40 mM NaCl, the number of binding sites increased. NaCl also altered the effects of Mg2+ and guanyl nucleotides. At low ionic strength, Mg2+ was necessary to observe binding. Guanine nucleotides modulated this binding by decreasing the affinity. At 40 mM NaCl, Mg2+ increased receptor number without altering affinity. Guanyl nucleotides modulated this binding by reducing the number of sites to that observed in the absence of Mg2+. At 150 mM NaCl, Mg2+ and guanine nucleotides had no effect. The results suggest the presence of two pools of human choriogonadotropin receptor in rat corpus luteum, one coupled to the guanine nucleotide regulatory protein (Ns) and being Mg2+ dependent and guanine nucleotide sensitive, and the other not coupled to Ns and being Mg2+ independent and guanine nucleotide insensitive.

Fast-phase transvascular fluid flux and the Fahraeus effect

1987 ◽  
Vol 62 (4) ◽  
pp. 1513-1520 ◽  
Author(s):  
W. N. Richardson ◽  
D. Bilan ◽  
M. Hoppensack ◽  
L. Oppenheimer
Keyword(s):  
Mechanical Properties ◽  
Slow Rate ◽  
Time Course ◽  
Rate Of Change ◽  
Distributed Model ◽  
Slow Phase ◽  
Fluid Flux ◽  
Fast Phase ◽  
Constant Rate ◽  
Fahraeus Effect

Transvascular fluid flux was induced in six isolated blood-perfused canine lobes by increasing and decreasing hydrostatic inflow pressure (Pi). Fluid flux was followed against the change in concentration of an impermeable tracer (Blue Dextran) measured directly with a colorimetric device. The time course of fluid flux was biphasic with an initial fast transient followed by a slow phase. Hematocrit changes unrelated to fluid flux occurred due to the Fahraeus effect, and their contribution to the total color signal was subtracted to determine the rate of fast fluid flux (Qf). Qf was related to Pi to derive fast-phase conductance (Kf). Slow-phase Kf was calculated from the constant rate of change of lobe weight. For a mean change in Pi of 7 cmH2O, 40% of the color signal was due to fluid flux. Fast- and slow-phase Kf's were 0.86 +/- 0.15 and 0.27 +/- 0.05 ml X min-1. cmH2O–1 X 100 g dry wt-1. The fast-phase Kf is smaller than that reported for plasma-perfused lobes. Possible explanations discussed are the nature of the perfusate, the mechanical properties of the interstitium, and the slow rate of rise of the driving pressure at the filtration site on the basis of a distributed model of pulmonary vascular compliance.

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