Maximal Ca2+ i stimulation of cardiac Na+/Ca2+ exchange requires simultaneous alkalinization and binding of PtdIns-4,5-P2 to the exchanger

2007 ◽  
Vol 388 (3) ◽  
pp. 281-288 ◽  
Author(s):  
Velia Posada ◽  
Luis Beaugé ◽  
Graciela Berberián
Keyword(s):  
De Novo ◽  
Regulatory Site ◽  
Synergistic Inhibition ◽  
Heart Sarcolemma ◽  
Intracellular Ca ◽  
Intracellular Medium ◽  
Inside Out ◽  
Total Membrane ◽  
Intracellular Alkalinization ◽  
Stimulation Of

Abstract Using bovine heart sarcolemma vesicles we studied the effects of protons and phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) on the affinity of the mammalian Na+/Ca2+ exchanger (NCX1) for intracellular Ca2+. By following the effects of extravesicular ligands in inside-out vesicles, their interactions with sites of NCX1 facing the intracellular medium were investigated. Two Na+-gradient-dependent fluxes were studied: Ca2+ uptake and Ca2+ release. PtdIns-4,5-P2 binding to NCX1 was investigated in parallel. Without MgATP (no ‘de novo’ synthesis of PtdIns-4,5-P2), alkalinization increased the affinity for Ca2+ and the PtdIns-4,5-P2 bound to NCX1. Vesicles depleted of phosphoinositides were insensitive to alkalinization, but became responsive following addition of exogenous PtdIns-4,5-P2 or PtdIns plus MgATP. Acidification reduced the affinity for Ca2+ ev; this was only partially reversed by MgATP, despite the increase in bound PtdIns-4,5-P2 to levels observed with alkalinization. Inhibition of Ca2+ uptake by increasing extravesicular [Na+] indicates that it is related to H+ i and Na+ i synergistic inhibition of the Ca2+ i regulatory site. Therefore, the affinity of the NCX1 Ca2+ i regulatory site for Ca2+ was maximal when both intracellular alkalinization and an increase in PtdIns-4,5-P2 bound to NCX1 (not just of the total membrane PtdIns-4,5-P2) occurred simultaneously. In addition, protons influenced the distribution, or the exposure, of PtdIns-4,5-P2 molecules in the surroundings and/or on the exchanger protein.

Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation

1995 ◽  
Vol 73 (01) ◽  
pp. 039-048 ◽  
Author(s):  
A Bierhaus ◽  
Ch J Hemmer ◽  
N Mackman ◽  
R Kutob ◽  
R Ziegler ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Falciparum Malaria ◽  
De Novo ◽  
Neutralizing Antibody ◽  
Mobility Shift ◽  
Before And After ◽  
Tf Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Antiparasitic Treatment ◽  
Stimulation Of

SummarySerum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.

Testosterone-augmented contractile responses to α1- and β1-adrenoceptor stimulation are associated with increased activities of RyR, SERCA, and NCX in the heart

2009 ◽  
Vol 296 (4) ◽  
pp. C766-C782 ◽  
Author(s):  
Sharon Tsang ◽  
Stanley S. C. Wong ◽  
Song Wu ◽  
Gennadi M. Kravtsov ◽  
Tak-Ming Wong
Keyword(s):  
Ventricular Myocytes ◽  
Left Ventricular ◽  
Contractile Responses ◽  
Adrenoceptor Antagonist ◽  
Cardiac Contractile ◽  
Plasmic Reticulum ◽  
Adrenoceptor Agonist ◽  
Intracellular Ca ◽  
Developed Pressure ◽  
Stimulation Of

We hypothesized that testosterone at physiological levels enhances cardiac contractile responses to stimulation of both α1- and β1-adrenoceptors by increasing Ca2+ release from the sarcoplasmic reticulum (SR) and speedier removal of Ca2+ from cytosol via Ca2+-regulatory proteins. We first determined the left ventricular developed pressure, velocity of contraction and relaxation, and heart rate in perfused hearts isolated from control rats, orchiectomized rats, and orchiectomized rats without and with testosterone replacement (200 μg/100 g body wt) in the presence of norepinephrine (10−7 M), the α1-adrenoceptor agonist phenylephrine (10−6 M), or the nonselective β-adrenoceptor agonist isoprenaline (10−7 M) in the presence of 5 × 10−7 M ICI-118,551, a β2-adrenoceptor antagonist. Next, we determined the amplitudes of intracellular Ca2+ concentration transients induced by electrical stimulation or caffeine, which represent, respectively, Ca2+ release via the ryanodine receptor (RyR) or releasable Ca2+ in the SR, in ventricular myocytes isolated from the three groups of rats. We also measured 45Ca2+ release via the RyR. We then determined the time to 50% decay of both transients, which represents, respectively, Ca2+ reuptake by sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and removal via the sarcolemmal Na+/Ca2+ exchanger (NCX). We correlated Ca2+ removal from the cytosol with activities of SERCA and its regulator phospholamban as well as NCX. The results showed that testosterone at physiological levels enhanced positive inotropic and lusitropic responses to stimulation of α1- and β1-adrenoceptors via the androgen receptor. The increased contractility and speedier relaxation were associated with increased Ca2+ release via the RyR and faster Ca2+ removal out of the cytosol via SERCA and NCX.

Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae

2000 ◽  
Vol 279 (1) ◽  
pp. C274-C280 ◽  
Author(s):  
Andrew P. Krivoshik ◽  
Lloyd Barr
Keyword(s):  
Myosin Light Chain ◽  
Smooth Muscles ◽  
Cytosolic Calcium ◽  
Myosin Phosphorylation ◽  
Primary Signal ◽  
Intracellular Ca ◽  
Phosphorylation Cascade ◽  
Muscle Myosin ◽  
Light Flashes ◽  
Stimulation Of

In the rat sphincter pupillae, as in other smooth muscles, the primary signal transduction cascade for agonist activation is receptor → G protein → phospholipase C → inositol trisphosphate → intracellular Ca2+concentration ([Ca2+]i) → calmodulin → myosin light chain kinase → phosphorylated myosin → force development. Light stimulation of isolated sphincters pupillae can be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMR ideal for testing models of regulation of smooth muscle myosin phosphorylation. We measured force and [Ca2+]iconcurrently in sphincter pupillae following stimulation by light flashes of varying duration and intensity. We sampled at unusually short (0.01–0.02 s) intervals to adequately test a PMR model based on the myosin phosphorylation cascade. We found, surprisingly, contrary to the behavior of intestinal muscle and predictions of the phosphorylation model, that during PMRs force begins to decay while [Ca2+]iis still rising. We conclude that control of contraction in the sphincter pupillae probably involves an inhibitory process as well as activation by [Ca2+]i.

scholarly journals Induction of dopa (3,4-dihydroxyphenylalanine) decarboxylase in blowfly integument by ecdysone. A demonstration of synthesis of the enzyme de novo

1975 ◽  
Vol 146 (1) ◽  
pp. 121-126 ◽  
Author(s):  
E G Fragoulis ◽  
C E Sekeris
Keyword(s):  
Enzyme Activity ◽  
Steroid Hormones ◽  
De Novo ◽  
Steroid Hormone ◽  
Dopa Decarboxylase ◽  
Double Labelling ◽  
Labelling Technique ◽  
Immune Precipitation ◽  
Enzyme Molecules ◽  
Stimulation Of

The activity of the enzyme dopa (3,4-dihydroxyphenylalanine) decarboxylase, present in the epidermis cells of blowfly larvae, increases during the late third instar under the influence of the steroid hormone, ecdysone. By using the double-labelling technique and immune precipitation with univalent antibody to dopa decarboxylase, we demonstrated that the increase in enzyme activity was due to a stimulation of synthesis of enzyme molecules de novo. In this respect, the action of ecdysone is similar to the action of other steroid hormones.

scholarly journals Nature of the stimulation of biogenesis of cholesterol in the liver by noradrenaline

1977 ◽  
Vol 162 (3) ◽  
pp. 493-499 ◽  
Author(s):  
R George ◽  
T Ramasarma
Keyword(s):  
Protein Synthesis ◽  
Adrenergic Receptors ◽  
De Novo ◽  
Direct Action ◽  
Time Intervals ◽  
Coa Reductase ◽  
Short Time ◽  
Stimulation Of

1. Administration of noradrenaline increased the incorporation of [1-14C]acetate into hepatic sterols and the activity of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase. 2. The stimulation was observed at short time-intervals with a maximum at 4h and was progressive with increasing concentrations of noradrenaline. 3. Protein synthesis de novo was a necessary factor for the effect. 4. The stimulatory effect was not mediated through the adrenergic receptors, but appears to involve a direct action of the hormone within the hepatocyte.

scholarly journals NEURONAL-GLIAL MEMBRANE CONTACTS DURING PESSIMAL ELECTRICAL STIMULATION

2020 ◽  
Vol 28 (3) ◽  
pp. 35-50
Author(s):  
Oleg S. Sotnikov ◽  
Svetlana S. Sergeeva ◽  
Tat'yana I. Vasyagina
Keyword(s):  
Electrical Stimulation ◽  
Gap Junctions ◽  
De Novo ◽  
Laboratory Rats ◽  
Transmission Electron ◽  
Layer Structures ◽  
Dense Substance ◽  
Membrane Contacts ◽  
Glial Membrane ◽  
Stimulation Of

After the creation of a method for obtaining inter-neuronal gap junctions in a nervous system devoid of glia, it is expedient to reproduce gap neuronal-glial contacts on a model that also contains hybrid neuronal-glial gap junctions, which, as you know, are functionally fundamentally different from inter-neuronal contacts. The experiments were carried out on the truncus sympathicus ganglia of laboratory rats using pessimal electrical stimulation and transmission electron microscopy. Electrical activation of ganglia with a frequency of up to 100 Hz revealed local and widespread variants of various neuronal-glial connections (contacts, bridges), fringed with peri-membrane filamentous proteins. They had a blurred veil that masked two-layer neuro-membranes. Some of the contacts resembled slit or dense 5-layer structures without a visible inter-neuronal slit, but with an extreme decrease in the thickness of the contact slit. The main result of the experiments was the formation, in addition to slotted, multiple septate (ladder) contacts. Relatively independent aggregates of the electron-dense substance of the septa were located inside the intercellular gaps, crossing both adjacent membranes, and, possibly, permeate of them. Near-membrane, poorly outlined pyramid-like protein cones associated with both cell membranes were also formed. Such membranes appeared to be dotted-dashed, that is, not continuous. A significant number of septic contact membranes had endocytic invaginations (invaginations) facing neuroplasm with pyramid-like marginal projections. All reactive altered structures that have arisen de novo are considered by the authors as developed under the influence of frequency electrical stimulation of denaturation and aggregation of intrinsic and perimembrane proteins.

scholarly journals EGF stimulates an increase in actin nucleation and filament number at the leading edge of the lamellipod in mammary adenocarcinoma cells

1998 ◽  
Vol 111 (2) ◽  
pp. 199-211 ◽  
Author(s):  
A.Y. Chan ◽  
S. Raft ◽  
M. Bailly ◽  
J.B. Wyckoff ◽  
J.E. Segall ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Leading Edge ◽  
Mammary Adenocarcinoma ◽  
Actin Dynamics ◽  
Actin Nucleation ◽  
Nucleation Sites ◽  
Nucleation Activity ◽  
Pointed End ◽  
Stimulation Of

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12+/−0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.

Stimulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by proteinase pretreatment

1982 ◽  
Vol 243 (3) ◽  
pp. C191-C195 ◽  
Author(s):  
K. D. Philipson ◽  
A. Y. Nishimoto
Keyword(s):  
Polymyxin B ◽  
Na Pump ◽  
Inside Out ◽  
Sarcolemmal Vesicles ◽  
Thiol Proteinase ◽  
Stimulation Of ◽  
Exchange Activity

The Na+-Ca2+ exchange activity of purified canine cardiac sarcolemmal vesicles can be strikingly stimulated if the vesicles are pretreated with a serine or thiol proteinase. The Km (Ca2+) for Na+i-dependent Ca2+ influx is reduced from 22.2 +/- 2.3 to 8.1 +/- 0.3 microM while Vmax is increased from 15.1 +/- 3.6 to 18.9 +/- 5.2 nmol Ca2+ . mg protein-1 . s-1. Na+o-dependent Ca2+ efflux is also stimulated by proteinase pretreatment although passive (Na+-independent) Ca2+ efflux from the sarcolemmal vesicles is unaffected. Proteinase treatment reduces the sensitivity of Na+-Ca2+ exchange to the inhibitors chlorpromazine and polymyxin B, but not to the inhibitor, palmitylcarnitine. Using a newly developed technique we are able to demonstrate that the Na+-Ca2+ exchange of inside-out sarcolemmal vesicles is being stimulated by proteinase treatment (Philipson, K. D., and A. Y. Nishimoto, J. Biol. Chem: 257, 5111-5117, 1982; this technique uses the ATP-dependent Na+ pump to preload only inside-out vesicles with Na+ prior to Na+-Ca2+ exchange). Right-side-out vesicles may also be stimulated.

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