Furosemide-sensitive thallium fluxes in smooth muscle of rabbit uterus

1983 ◽  
Vol 245 (6) ◽  
pp. F778-F783
Author(s):  
A. Johns ◽  
S. V. Cutshaw
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Rank Order ◽  
Chloride Concentration ◽  
Cation Binding ◽  
Exchange System ◽  
Total Uptake ◽  
Low Concentrations ◽  
Chloride Pump ◽  
High Concentrations

The furosemide-sensitive uptake of thallium represents approximately equal to 50% of the total uptake of thallium by rabbit uterus and requires Cl- and Na+. The furosemide-sensitive uptake of thallium is stimulated by other ions at low concentrations with the rank order Li+ greater than Tl+ greater than K+ = Rb+ greater than Cs+ and is inhibited by these ions at high concentrations with the rank order Tl+ greater than K+ = Rb+ greater than Cs+ greater than Li+, suggesting multiple cation binding sites on the carrier. Uptake of 36Cl- is inhibited by furosemide in the presence of ouabain. Thallium efflux and 36Cl efflux in the presence of ouabain is inhibited by furosemide. The chloride concentration regulates the proportion of thallium uptake that is ouabain sensitive and furosemide sensitive without altering the total uptake. It is suggested that the furosemide-sensitive uptake of thallium reflects a Na+-Cl- -K+ exchange system that could be classified as a cotransport or countertransport of any two of these ions and also could be the smooth muscle chloride pump.

scholarly journals Sugar Transport and Metal Binding in Yeast

1965 ◽  
Vol 49 (2) ◽  
pp. 235-246 ◽  
Author(s):  
Johnny van Steveninck ◽  
Aser Rothstein
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Equilibrium Distribution ◽  
Sugar Transport ◽  
Cation Binding ◽  
Facilitated Diffusion ◽  
Low Concentrations ◽  
Michaelis Constants ◽  
High Concentrations ◽  
Phosphoryl Groups

The uptake of sugars by yeast can be separated into two classes. The first involves the uptake of sorbose or galactose by starved cells, and the uptake of glucose by iodoacetate-poisoned cells. These uptakes do not involve any changes in Ni++- or Co++-binding by the cell surface, are not inhibited by Ni++, are inhibited by UO2++ in relatively high concentrations, are characterized by high Michaelis constants and low maximal rates and by a final equilibrium distribution of the sugars. The second involves the uptake of glucose in unpoisoned cells and galactose in induced cells. These uptakes are characterized by a reduction of Ni++- and Co++-binding, by a partial inhibition by Ni++, by an inhibition with UO2++ in relatively low concentrations, and by a low Km and a high Vm. In the case of galactose in induced cells, previous studies demonstrate that the sugar is accumulated against a concentration gradient. It is suggested that the first class of uptakes involves a "facilitated diffusion" via a relatively non-specific carrier system, but the second represents an "uphill" transport involving the highly specific carriers, and phosphoryl groups (cation-binding sites) of the outer surface of the cell membrane.

Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

1986 ◽  
Vol 55 (01) ◽  
pp. 136-142 ◽  
Author(s):  
K J Kao ◽  
David M Shaut ◽  
Paul A Klein
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Platelet Binding ◽  
Functional Involvement ◽  
Thrombin Activation ◽  
Platelet Aggregates ◽  
High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin

2000 ◽  
Vol 279 (6) ◽  
pp. C1838-C1846 ◽  
Author(s):  
Beate Illek ◽  
Mike E. Lizarzaburu ◽  
Vivien Lee ◽  
Michael H. Nantz ◽  
Mark J. Kurth ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Rank Order ◽  
Molecular Structures ◽  
Structural Determinants ◽  
Chloride Currents ◽  
Binding Characteristics ◽  
Low Concentrations ◽  
Current Block ◽  
Maximal Binding ◽  
High Concentrations

Apigenin (4′,5,7-trihydroxyflavone) is an activator of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl−currents across epithelia at low concentrations and a blocker at high concentrations. We determined the roles of structural components of apigenin for both stimulation and block of Cl−currents across Calu-3 epithelia. The half-maximal binding affinity of apigenin for current stimulation ( Ks) was 9.1 ± 1.3 μM, and the rank-order of molecular structures was 7-hydroxyl > pyrone = 4′-hydroxyl > 5-hydroxyl. Both the 7-hydroxyl and the 4′-hydroxyl served as H-bond acceptors, whereas the 5-hydroxyl was an H-bond donor. The half-maximal binding affinity of apigenin during current block was 74 ± 11 μM. Blocked Cl−currents were structurally determined by 7-hydroxyl = 4′-hydroxyl > pyrone > 5-hydroxyl. Prestimulation of tissues with forskolin significantly affected activation kinetics and binding characteristics. After forskolin stimulation, Kswas 4.1 ± 0.9 μM, which was structurally determined by pyrone > all hydroxyls > single hydroxyls. In contrast, block of Cl−current by apigenin was not affected by forskolin stimulation. We conclude that apigenin binds to a stimulatory and an inhibitory binding site, which are distinguished by their affinities and the molecular interactions during binding.

Analysis of purine- and pyrimidine-induced vascular responses in the isolated rat cerebral arteriole

2001 ◽  
Vol 280 (2) ◽  
pp. H767-H776 ◽  
Author(s):  
Tetsuyoshi Horiuchi ◽  
Hans H. Dietrich ◽  
Shinichiro Tsugane ◽  
Ralph G. Dacey
Keyword(s):  
Smooth Muscle ◽  
Pyridoxal Phosphate ◽  
Disulfonic Acid ◽  
Dependent Manner ◽  
Cerebral Microvessels ◽  
Vascular Responses ◽  
Single Receptor ◽  
Low Concentrations ◽  
High Concentrations ◽  
Cerebral Arteriole

Effects of extraluminal UTP were studied and compared with vascular responses to ATP and its analogs in rat cerebral-penetrating arterioles. UTP, UDP, 2-methylthio-ATP, and α,β-methylene-ATP dilated arterioles at the lowest concentration and constricted them at high concentrations. Low concentrations of ATP dilated the vessels; high concentrations caused a biphasic response, with transient constriction followed by dilation. Endothelial impairment inhibited ATP- and UTP-mediated dilation and potentiated constriction to UTP but not to ATP. ATP- and 2-methylthio-ATP- but not UTP-mediated constrictions were inhibited by desensitization with 10−6M α,β-methylene-ATP or 3 × 10−6M pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). PPADS at 10−4M abolished the UTP-mediated constriction and induced vasodilation in a dose-dependent manner but did not affect the dilation to ATP. These results suggest that in rat cerebral microvessels 1) ATP and 2-methylthio-ATP induce transient constriction via smooth muscle P2X1receptors in the cerebral arteriole, 2) UTP stimulates two different classes of P2Yreceptors, resulting in constriction (smooth muscle P2Y4) and dilation (possibly endothelial P2Y2), and 3) ATP and UTP produce dilation by stimulation of a single receptor (P2Y2).

scholarly journals Calcitonin Gene-Related Peptide (Cgrp), Adrenomedullin (Am), Amylin, And Calcitonin (Ct) Receptors And Overlapping Biological Actions

2001 ◽  
Vol 1 ◽  
pp. 4-4 ◽  
Author(s):  
J.A. Fischer ◽  
W. Born ◽  
R. Muff
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Glycogen Synthesis ◽  
Orphan Receptor ◽  
Cgrp Receptor ◽  
Low Concentrations ◽  
Ring Structures ◽  
High Concentrations ◽  
Receptor Activity Modifying Proteins ◽  
Biological Actions

CGRP, AM, amylin, and CT have in common N-terminal 6-7 amino acid ring structures linked by disulfide bridges and amidated C-termini required for biological activity. For the related bioactive peptides, receptor-binding sites linked to cAMP stimulation and to a lesser extent to the phospholipase C signaling pathway have been identified in tissue specific manner. The highest density of CGRP receptors has been recognized in the cerebellum and the spinal cord. There photoaffinity-labeled N-glycosylated 60,000 and 54,000 Mr proteins are converted to 46,000 and 41,000 Mr components following endoglycosidase F/N-glycosidase F treatment. The same proteins were specifically labeled with [125I]-hCGRP-I(1-37) and -(8-37). Some cross-reaction between the CGRP receptor and AM was evident whereas amylin and CT were only recognized at over 10-7 M. A different AM receptor localized predominantly in the lung recognized CGRP at low, and amylin and calcitonin at equally high concentrations. CT receptor binding sites have been identified in osteoclasts and in the periventricular region of the brain. They cross-reacted with amylin at low concentrations and with CGRP and AM at over 10-7 M. Amylin receptor binding sites cross-reacting with salmon CT and CGRP but not with hCT and adrenomedullin to any great extent were originally described by Sexton in the nucleus accumbens and may represent a second CGRP receptor. The structure of a CT receptor was elucidated by the group of Goldring in 1991 through molecular cloning, and of a 60% homologous human CT receptor-like receptor (CRLR) shortly thereafter here. The latter was an orphan receptor until the discovery of the receptor-activity-modifying proteins (RAMP) by Foord which upon coexpression yield a CGRP receptor with RAMP1 and an AM receptor with RAMP2. Coexpression of the hCT receptor isotype 2 revealed a CGRP/amylin receptor with RAMP1 and an amylin receptor isotype with RAMP3. The CRLR/RAMP1 receptor antagonized by CGRP(8-37) corresponds to the CGRP1 receptor defined by Quirion, whereas his CGRP2 receptor remains to be identified. Another CGRP receptor isotype remains to be discovered in the cerebellum with no detectable CRLR encoding mRNA. Overlapping biological actions include inhibition of bone resorption obtained predominantly with CT, but also at high concentrations with CGRP, AM, and amylin. CGRP and AM are potent vasodilators, an effect shared with CT at pharmacological concentrations. Biological actions of amylin include suppression of insulin secretion, stimulation of glycogenolysis and inhibition of glycogen synthesis. In conclusion, the hCT2 receptor or the CRLR are associated with one of three RAMPs to bind to CGRP, AM, or amylin.

scholarly journals Mode of Action of the Bordetella BvgA Protein: Transcriptional Activation and Repression of the Bordetella bronchiseptica bipA Promoter

2005 ◽  
Vol 187 (18) ◽  
pp. 6290-6299 ◽  
Author(s):  
Meenu Mishra ◽  
Rajendar Deora
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Expression Profiles ◽  
Regulation Of Gene Expression ◽  
Binding Assays ◽  
Specific Expression ◽  
Low Concentrations ◽  
Complex Regulation ◽  
High Concentrations

ABSTRACT The Bordetella BvgAS signal transduction system controls the transition among at least three known phenotypic phases (Bvg+, Bvgi, and Bvg−) and the expression of a number of genes which have distinct phase-specific expression profiles. This complex regulation of gene expression along the Bvg signaling continuum is best exemplified by the gene bipA, which is expressed at a low level in the Bvg+ phase, at a maximal level in the Bvgi phase, and at undetectable levels in the Bvg− phase. The bipA promoter has multiple BvgA binding sites which play distinct regulatory roles. We had previously speculated that the expression profile of bipA is a consequence of the differential occupancy of the various BvgA binding sites as a result of variation in the levels of phosphorylated BvgA (BvgA-P) inside the cell. In this report, we provide in vitro evidence for this model and show that bipA expression is activated at low concentrations of BvgA-P and is repressed at high concentrations. By using independent DNA binding assays, we demonstrate that under activating conditions there is a synergistic effect on the binding of BvgA and RNA polymerase (RNAP), leading to the formation of open complexes at the promoter. We further show that, under in vitro conditions, when bipA transcription is minimal, there is competition between the binding of RNAP and BvgA-P to the bipA promoter. Our results show that the BvgA binding site IR2 plays a central role in mediating this repression.

scholarly journals Influx of calcium, strontium, and barium in presynaptic nerve endings.

1982 ◽  
Vol 79 (6) ◽  
pp. 1065-1087 ◽  
Author(s):  
D A Nachshen ◽  
M P Blaustein
Keyword(s):  
Divalent Cation ◽  
Binding Sites ◽  
Divalent Cations ◽  
Ion Selectivity ◽  
Permeability Ratio ◽  
Low Concentrations ◽  
Slow Channel ◽  
Channel Selectivity ◽  
High Concentrations ◽  
Two Phases

Depolarization-induced (potassium-stimulated) influx of 45Ca, 85Sr, and 133Ba was measured in synaptosomes prepared from rat brain. There are two phases of divalent cation entry, "fast" and "slow;" each phase is mediated by channels with distinctive characteristics. The fast channels inactivate (within 1 s) and are blocked by low concentrations (less than 1 micro M) of La. The slow channels do not inactivate (within 10 s), and are blocked by high concentrations (greater than 50 micro M) of La. Divalent cation influx through both channels saturates with increasing concentrations of permeant divalent cation; in addition, each permeant divalent cation species competitively blocks the influx of other permeant species. These results are consistent with the presence of "binding sites" for divalent cations in the fast and slow channels. The Ca:Sr:Ba permeability ratio, determined by measuring the influx of all three species in triple-label experiments, was 6:3:2 for the fast channel and 6:3:1 for the slow channel. A simple model for ion selectivity, based on the presence of a binding site in the channel, could account well for slow and, to some extent, for fast, channel selectivity data.

scholarly journals Effects of divalent cations on dynein cross bridging and ciliary microtubule sliding.

1979 ◽  
Vol 80 (3) ◽  
pp. 573-588 ◽  
Author(s):  
N C Zanetti ◽  
D R Mitchell ◽  
F D Warner
Keyword(s):  
Binding Sites ◽  
Divalent Cations ◽  
Substantial Reduction ◽  
Cation Binding ◽  
Cation Concentration ◽  
Dependent Relationship ◽  
Effective Inhibition ◽  
High Concentrations ◽  
Cross Bridges ◽  
Dose Dependent

We recently demonstrated that addition of the divalent cation Mg++ to demembranated cilia causes the dynein arms to attach uniformly to the B subfibers. We have now studied the dose-dependent relationship between Mg++ or Ca++ and dynein bridging frequencies and microtubule sliding in cilia isolated from Tetrahymena. Both cations promote efficient dynein bridging. Mg++-induced bridges become saturated at 3 mM while Ca++-induced bridges become saturated at 2 mM. Double reciprocal plots of percent bridging vs. the cation concentration (0.05-10 mM) suggest that bridging occurs in simple equilibrium with the cation concentration. When microtubule sliding (spontaneous disintegration in 40 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), 0.1 mM ATP at pH 7.4) is assayed (A350 nm) relative to the Mg++ or Ca++ concentration, important differential effects are observed. 100% Disintegration occurs in 0.5-2 mM Mg++ and the addition of 10 mM Mg++ does not inhibit the response. The addition of 0.05-10 mM Ca++ to cilia reactivated with 0.1 mM ATP causes a substantial reduction in disintegration at low Ca++ concentrations and complete inhibition at concentrations greater than 3 mM. When Ca++ is added to cilia reactivated with 2 mM Mg++ and 0.1 mM ATP, the percent disintegration decreases progressively with the increasing Ca++ concentration. The addition of variable concentrations of Co++ to Mg++-activated cilia causes a similar but more effective inhibition of the disintegration response. These observations, when coupled with the relatively high concentrations of Ca++ or Co++ needed to inhibit disintegration, suggest that inhibition results from simple competition for the relevant cation-binding sites and thus may not be physiologically significant. The data do not yet reveal an interpretable relationship between percent disintegration, percent dynein bridging, and percent ATPase activity of both isolated dynein and whole cilia. However, they do illustrate that considerable (sliding) disintegration (60%) can occur under conditions that reveal only 10-15% attached dynein cross bridges.

Effects of amino acids on Thiobacillus acidophilus. II. Threonine deaminase

1980 ◽  
Vol 26 (3) ◽  
pp. 385-388 ◽  
Author(s):  
Gérald Proteau ◽  
Marvin Silver
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Ammonium Sulfate ◽  
Active Site ◽  
Binding Sites ◽  
Threonine Deaminase ◽  
Low Concentrations ◽  
High Concentrations ◽  
Thiobacillus Acidophilus ◽  
Optimal Ph

Biosynthetic L-threonine deaminase was partially purified 73-fold with a 60% recovery from Thiobacillus acidophilus by ammonium sulfate fractionation and by Sepharose 6B-C1 chromatography. The optimal pH for enzyme activity was between 9.0 and 10.0 and no optimal pH shift was observed in the presence of L-isoleucine, an inhibitor. The enzyme was effectively inhibited by L-isoleucine and showed homotropic interaction only in the presence of L-isoleucine.Kinetic studies indicate that there are at least two threonine binding sites and at least two isoleucine binding sites. The Km for threonine is 2.5 × 10−3 M. The inhibition due to isoleucine is reversed by low concentrations of L-valine. L-Valine at high concentrations acts as a substrate analogue and competitively inhibits L-threonine binding at the active site; the K1 is 1.6 × 10−2 M.

Phenylephrine contracts rat tail artery by one electromechanical and three pharmacomechanical mechanisms

1995 ◽  
Vol 268 (1) ◽  
pp. H74-H81 ◽  
Author(s):  
X. L. Chen ◽  
C. M. Rembold
Keyword(s):  
Smooth Muscle ◽  
Isometric Force ◽  
Rat Tail Artery ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Tail Artery ◽  
Low Concentrations ◽  
Voltage Dependent ◽  
High Concentrations ◽  
Rat Tail

There are at least four mechanisms hypothesized to account for excitation-contraction coupling in arterial smooth muscle. 1) Contractile agonists and changes in extracellular [K+] ([K+]o) induce contraction by depolarization, which increases Ca2+ influx; this is the only mechanism involving a change in membrane potential (Em). 2) Contractile agonists release Ca2+ from the intracellular Ca2+ store. 3) Contractile agonists increase Ca2+ influx without changing Em either by activating voltage-dependent L-type Ca2+ channels or by opening other Ca(2+)-permeable channels. 4) Contractile agonists increase intracellular Ca2+ ([Ca2+]i) sensitivity of force; this is the only mechanism that does not involve changes in [Ca2+]i. Each of these mechanisms has been demonstrated in intact, skinned, or dissociated smooth muscle preparations. However, these four mechanisms have not been compared in the same preparation. The goal of this study was to determine which of these four contractile mechanisms are physiologically relevant in the intact rat tail artery. We stimulated deendothelialized rat tail artery with phenylephrine and high [K+]o. We then measured Em with microelectrodes, [Ca2+]i with fura 2, and isometric force with a strain gauge transducer. We find that all four mechanisms contributed to phenylephrine-induced rat tail artery contraction. The majority of phenylephrine-induced contraction was caused by depolarization and by increases in the [Ca2+]i sensitivity of force. Low concentrations of phenylephrine also increased [Ca2+] independent of changes in Em, potentially by increases in Ca2+ influx. Release of Ca2+ from intracellular stores was only observed with high concentrations of phenylephrine. Smooth muscle appears to invoke multiple mechanisms for excitation-contraction coupling.

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