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E Jaimovich

Publications and source records attributed to E Jaimovich.

At least 37 records · Page 2Linked to original sources
PubMed1990

Calcium release modulated by inositol trisphosphate in ruptured fibers from frog skeletal muscle.

To investigate the effect of inositol 1,4,5-trisphosphate on calcium release, we used fiber bundles of frog sartorius muscle mechanically permeabilized by a scratching procedure, and we detected increments in calcium concentration by measuring aqueorin light signals. Submicromolar concentrations of inositol 1,4,5-trisphosphate induced fast calcium-release signals, with a half time to peak of 60 ms or less. Similar responses were elicited by caffeine. The calcium-release signal induced by inositol 1,4,5-trisphosphate occurred at pCa values of 7 or lower, and the dose-response curve depended on the ionic composition of the incubation solution. Lower inositol 1,4,5-trisphosphate concentrations were needed to induce release when incubation solutions of ionic composition expected to depolarize the transverse tubule membrane were used. Inositol 1,4,5-trisphosphate was more effective than inositol 1,3,4-trisphosphate, inositol 1,4,5,6-tetrakisphosphate, and inositol 1,4-bisphosphate. The effect of inositol 1,4,5-trisphosphate was synergistic with that of caffeine, and was not inhibited by heparin. These results, by showing directly that at resting calcium levels inositol 1,4,5-trisphosphate elicited calcium release, are consistent with a role for inositol 1,4,5-trisphosphate as a chemical modulator in excitation/contraction coupling in skeletal muscle.

Aequorin↗
PubMed1989

Inositol trisphosphate and excitation-contraction coupling in skeletal muscle.

The role of inositol trisphosphate as a chemical messenger in excitation-contraction coupling is discussed, both in terms of positive and negative results. The evidence presented includes experiments on the effect of inositol trisphosphate in intact and skinned fibers, in calcium release from isolated sarcoplasmic reticulum vesicles, in activation of single calcium release channels incorporated in planar bilayers, and biochemical experiments that have established the presence of all the intermediate steps involved in the metabolism of phosphoinositides, both in intact muscle and in isolated membranes. From these results, it is clear that a role for inositol triphosphate in skeletal muscle function is highly likely; whether this molecule is the physiological messenger in excitation-contraction coupling remains to be established.

Animals↗
PubMed1988

Use of selective toxins to separate surface and tubular sodium currents in frog skeletal muscle fibers.

The interaction between toxin gamma from the venom of the scorpion Tityus serrulatus and sodium channels in skeletal muscle membranes from the frog Caudiverbera caudiverbera was studied. Sodium current from cut sartorius muscle fibers is a complex signal in which early and late components are difficult to separate. External application of Tityus gamma toxin initially blocked the early component in a voltage-dependent manner. Longer exposure to the toxin induced a complete blockade of the two components of the inward current. Application of tetrodotoxin to fibers pretreated with Tityus toxin at submaximal concentrations allowed the observation of the two distinct components of the inward current. Binding of 125I-labelled toxin to highly purified membrane fractions from the same muscle was used to establish the presence of high affinity receptors both in the transverse-tubular and in the surface membrane.

Animals↗
PubMed1988

Calcium modulation of phosphoinositide kinases in transverse tubule vesicles from frog skeletal muscle.

Highly purified transverse tubule membranes isolated from frog skeletal muscle phosphorylate phosphatidylinositol to phosphatidylinositol 4-phosphate and phosphatidylinositol (4,5)-bisphosphate. The two phosphorylation reactions have different calcium requirements. Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-phosphate, which takes place in both isolated transverse tubules and sarcoplasmic reticulum membrane, is independent of calcium in a range of concentrations from 10(-9) to 10(-6) M, and is progressively inhibited to 10% of the maximal values by increasing calcium to 10(-4) M or higher (K0.5 = 5 X 10(-6) M). In contrast, phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol (4,5)-bisphosphate, a reaction exclusively present in transverse tubule membranes, is maximal at calcium concentrations higher than 2 X 10(-6) M and decreases to 30% of maximal values at calcium concentrations of 2 X 10(-7) M or lower (K0.5 = 10(-6) M). Unlike frog membranes, transverse tubules from rabbit muscle need exogenous phosphatidylinositol 4-phosphate in order to produce the bisphosphate derivative in the same range of calcium concentrations. Inositol (1,4,5)-trisphosphate has been proposed recently as a chemical messenger in excitation-contraction coupling in skeletal muscle. Calcium regulation of the synthesis of phosphatidylinositol (4,5)-bisphosphate, the membrane-bound precursor of inositol (1,4,5)-trisphosphate, might have physiological implications regarding modulation of excitation-contraction coupling by intracellular calcium levels.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

Inositol (1,4,5)-trisphosphate activates a calcium channel in isolated sarcoplasmic reticulum membranes.

Sarcoplasmic reticulum membrane vesicles isolated from frog skeletal muscle display high conductance calcium channels when fused into phospholipid bilayers. The channels are selective for calcium and barium over Tris. The fractional open time was voltage-independent (-40 to +25 mV), but was steeply dependent on the free cis [Ca2+] (P0 = 0.02 at 10 microM cis Ca2+ and 0.77 at 150 microM Ca2+; estimated Hill coefficient: 1.6). Addition of ATP (1 mM; cis) further increased P0 from 0.77 to 0.94. Calcium activation was reversed by addition of EGTA to the cis compartment. Magnesium (2 mM) increased the frequency of rapid closures and 8 mM magnesium decreased the current amplitude from 3.4 to 1.2 pA at 0 mV, suggesting a reversible fast blockade. Addition of increasing concentrations of inositol (1, 4, 5)-triphosphate (cis), increased P0 from 0.10 +/- 0.01 (mean +/- SEM) in the control to 0.85 +/- 0.02 at 50 microM in an approximately sigmoidal fashion, with an apparent half-maximal activation at 15 microM inositol (1, 4, 5)-trisphosphate in the presence of 40 microM cis Ca2+. Lower concentrations of this agonist were required to produce a significant increase in P0 when 10 microM or less cis Ca2+ were used. The channel was blocked by the addition to the cis compartment of either 0.5 mM lanthanum, 0.5 microM ruthenium red, or 200 nM ryanodine, all known inhibitors of Ca2+ release from sarcoplasmic reticulum vesicles. These results demonstrate the presence of calcium channels in the sarcoplasmic reticulum from frog skeletal muscle with a pharmacological profile consistent with a role in excitation contraction coupling and with the hypothesis that inositol ( 1,4,5)-trisphosphate is a physiological agonist in this process.

Animals↗
PubMed1986

Phosphorylation of phosphatidylinositol by transverse tubule vesicles and its possible role in excitation-contraction coupling.

Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-monophosphate and to phosphatidylinositol 4,5-bisphosphate was demonstrated in transverse-tubule membranes isolated from frog skeletal muscle using [gamma-32P]ATP as substrate. At millimolar concentrations of Mg2+ both phosphorylation reactions were completed within 15 s at 25 degrees C. Isolated sarcoplasmic reticulum vesicles phosphorylated phosphatidylinositol to phosphatidylinositol 4-phosphate with a lower specific activity than the transverse tubules, and lacked the ability to produce phosphatidylinositol 4,5-bisphosphate. These findings show, for the first time, that isolated transverse-tubule membranes carry out one of the steps required to sustain a role for inositol trisphosphate as the physiological messenger in excitation-contraction coupling in skeletal muscle. The finding that 0.5 mM tetracaine apparently inhibits the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate also supports a role for these intermediates in excitation-contraction coupling.

Animals↗
PubMed1986

Sodium pathway markers in normal and kindled frog brains.

The present report evaluates Na,K-ATPase activity as well as Na channel levels in the frog telencephalon after kindling, i.e. the acquisition of an epileptic focus through localized low-voltage electrical stimulation of one hemisphere. K-dependent phosphatase activity and binding of tritiated ouabain were measured, revealing no change in Na,K-ATPase activity 14 h after the last seizure. Na channels were measured by binding assays using a tritiated ethylenediamine tetrodotoxin derivative. Na channels were reduced in kindled brain as compared to controls.

4-Nitrophenylphosphatase↗
PubMed1986

Tetrodotoxin-sensitive sodium channels in a continuously cultured cell line derived from the adult rat cerebellum.

A method to establish continuously cultured cell lines from adult cerebellar cortex is reported. Clones (prepared by this procedure) were isolated from cerebellar established cultures at the 25th passage and after 15 months in vitro. One clone (UCHCC1) was maintained in culture and studied while the others were frozen. The cerebellar cell line UCHCC1 retained a neuronal-like morphology; the addition of dimethylsulfoxide (DMSO) to the culture medium elicited a reproducible morphological 'differentiation' event, characterized mainly by process extension. In 'differentiated' cells, veratridine significantly increased the uptake of 22Na. Such enhanced uptake was blocked by tetrodotoxin (TTX) with a half-maximal inhibitory concentration of 0.9 nM. Binding of an [3H]ethylenediamine derivative of TTX ([3H]en-TTX) to the microsomal fraction prepared from same DMSO-treated cells, showed a single class of receptors with a maximal binding (Bmax) of 173 fmol/mg protein and a Kd of 1.1 nM. Thyroid UCHT1 cells and 'undifferentiated' (cultured without DMSO) cerebellar cells, did not show significant effects of veratridine on 22Na-uptake, or [3H]en-TTX binding. The 'differentiated' nerve-like properties, induced by appropriate environmental manipulation, demonstrate the usefulness of cerebellar UCHCC1 cells as a model system for the developing central neuron. On the other hand, the novel transforming procedure opens new possibilities for obtaining permanent cell lines from other regions of the adult CNS.

Animals↗
PubMed1986

Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation.

Transverse tubule vesicles were isolated from frog skeletal muscle by a procedure initially described by Rosemblatt et al. (J. Biol. Chem. 256, 8140-8148 (1981)) and later modified by Hidalgo et al. (J. Biol. Chem. 258, 13937-13945 (1983]. A large fraction of the isolated vesicles (80-90%) were sealed, as indicated by the detergent induced increase in (Na+ + K+)-ATPase activity and ATP-dependent ouabain binding. To determine the orientation of the sealed vesicles binding of digoxin, a lipid soluble derivative of ouabain, was measured. The same values of ATP-dependent digoxin binding were found with or without detergents, indicating that all the vesicles that are sealed have the ATP site accessible, and hence are sealed with the cytoplasmic side-out (inside-out orientation). The transverse tubule preparation isolated from frog muscle is highly purified, as indicated by its cholesterol content and its (Na+ + K+)-ATPase activity; negligible contamination with sarcoplasmic reticulum was observed, as indicated by the protein composition and the lack of measurable Ca2+-ATPase activity of the isolated transverse tubules. High initial rates of Mg2+-ATPase activity were found, with the peculiar property of being inhibited during the course of the reaction. Addition of lysophosphatidylcholine or saponin partially prevented the inhibition of Mg2+-ATPase activity during the reaction.

Animals↗
PubMed1986

Ion pathways in transverse tubules. Quantification of receptors in membranes isolated from frog and rabbit skeletal muscle.

The presence of four cation pathways in membrane vesicles isolated from transverse tubules of frog and rabbit skeletal muscle was studied by measuring binding of specific blockers. Transverse tubules purified from frog muscle have a maximal binding capacity for [3H]nitrendipine (a marker for voltage-dependent calcium channels) of 130 pmol/mg of protein; this binding is strongly dependent on temperature and, at 37 degrees C, on the presence of diltiazem. Receptors for [3H]ethylenediamine tetrodotoxin (a marker for voltage-dependent sodium channels) and for 125I-labeled alpha-bungarotoxin (a marker for acetylcholine-mediated channels) showed maximal binding values of about 5 pmol/mg. The number of sodium-pumping sites in the isolated tubule vesicles, inferred from [3H]ouabain binding, was 215 pmol/mg. The high purity of this preparation makes feasible the use of these values as a criterion to judge the degree of purity of isolated preparations, and it allows investigation of transverse tubule contamination in other muscle membrane fractions.

Acetylcholine↗
PubMed1983

The Na+ channel in mammalian cardiac cells. Two kinds of tetrodotoxin receptors in rat heart membranes.

The properties of interaction of both tetrodotoxin (TTX) and tritiated ethylenediamine tetrodotoxin [3H] en-TTX) were studied in rat heart membranes at different stages of development and in cultured cells. Studies by electrophysiology and by 22Na+ flux measurements on cardiac cultured cells indicate that the functional form of the Na+ channel is of low affinity for TTX (250-700 nM). Binding experiments (bioassay and [3H]en-TTX binding) on cultured cardiac cells from newborn rats indicate the presence of both high and low affinity binding sites for TTX with dissociation constants (Kd) of 1.6 and 135 nM, respectively. On homogenates of hearts taken just after birth, [3H]en-TTX binding reveals no high affinity binding site for TTX but the presence of a low affinity binding site with a Kd of 125 nM. This result was confirmed by kinetic studies and competition experiments. Conversely, binding studies on homogenates and extensively purified membranes from adult ventricles reveal the presence of both high and low affinity binding sites for TTX with Kd values of 1.5 and 170 nM, respectively. The maximum binding capacity for the low affinity binding sites is 45 times higher than that of the high affinity binding sites. High affinity sites do not exist at the fetal stage or at birth, but after 5 days their number gradually increases to reach a maximum level around 45 days after birth. Conversely, the number of low affinity binding sites is essentially invariant between birth and adulthood. Monolayers of cardiac cells from hearts at 2 days after birth which have no high affinity TTX-binding sites in vivo develop both high and low affinity binding sites for TTX in vitro. The results presented here are the first direct demonstration of the coexistence in rat heart plasma membrane of two families of binding sites for TTX.

Animals↗
PubMed1983

[3H]nitrendipine receptors in skeletal muscle.

The richest source of receptors for the organic calcium channel blocker [3H]nitrendipine in muscle is the transverse tubule membrane. The tubular membrane preparation binds [3H]nitrendipine with a high affinity and has a very high number of [3H]nitrendipine binding sites. For example, for the transverse tubule membrane preparation from rabbit muscle, the dissociation constant of the nitrendipine-receptor complex is 1.8 +/- 0.3 nM and the maximum binding capacity Bmax = 50 +/- 6 pmol/mg of protein. Similar results have been found with a membrane preparation from frog muscle. The dissociation constant found at equilibrium is near that determined from the ratio of rate constants for association (kappa 1) and dissociation (kappa-1). Binding of [3H] nitrendipine is pH-dependent and reveals the presence of an essential ionizable group with a pK of 5.4 on the nitrendipine receptor. The binding is destroyed by proteases showing that the receptor is a protein. Three different classes of Ca2+ channel blockers inhibit [3H]nitrendipine to its specific site. (i) The dihydropyridine analogs of nitrendipine which are competitive inhibitors of [3H]nitrendipine. These molecules form tight complexes with the nitrendipine receptor with dissociation constants between 1.4 and 4.0 nM. (ii) Other antiarrhythmic molecules like verapamil, amiodarone, bepridil, and F13004 which are noncompetitive inhibitors of [3H]nitrendipine binding with dissociation constants between 0.2 and 1 microM. (iii) Divalent cations like Ni2+, Co2+, Mn2+, or Ca2+ which are noncompetitive inhibitors of [3H]nitrendipine binding with the following rank order of potency: Ni+ (K0.5 = 1.8 mM) greater than Co2+ (K0.5 = 2.7 mM) greater than Mn2+ (K0.5 = 4.8 mM) greater than Ca2+ (K0.5 = 65 mM).

Animals↗
PubMed1983

Differences in the properties of Na+ channels in muscle surface and T-tubular membranes revealed by tetrodotoxin derivatives.

The effect of ethylenediamine derivatives of tetrodotoxin (enTTXI and enTTXII) on frog skeletal muscle was studied both electrophysiologically and biochemically. Electrophysiological experiments with one of these molecules (enTTXI) showed that the concentrations needed to block the early phase of the inward sodium current (K0.5 = 7 nM) are much lower than those needed to block the late phase of inward current or muscle contraction (K0.5 = 40 nM). Conversely, tubular Na+ channels are more sensitive to enTTXII than are surface Na+ channels. Toxin binding to isolated muscle membranes was studied using 3H-enTTXI and 3H-enTTXII. The first derivative (3H-enTTXI) has a higher affinity Kd = 8 nM) for Na+ channels in the surface membrane than for Na+ channels in the T-tubular membrane (Kd greater than 20 nM). In contrast 3H-enTTXII has a higher affinity for the tubular Na+ channel (Kd = 0.2 nM) than for the receptor in surface membranes (Kd = 4nM). We conclude that Na+ channels in muscle surface and T-tubular membranes have different toxin-binding properties, which must reflect a difference in molecular structure.

Animals↗
PubMed1982

Lipid monolayer expansion by calcium-chlorotetracycline at the air/water interface and, as inferred from cell shape changes, in the human erythrocyte membrane.

Chemically induced shape changes of the human erythrocyte may result from cell membrane bending by surface tension changes at the lipid bilayer (Evans. E.A. (1974( Biophys. J. 14, 923-931) implicating differential expansion of the monolayers coupled to form the red cell membrane (Sheetz, M.P. and Singer, S.J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4457-4461). Interacting with calcium, the antibiotic chlorotetracycline (CTC) transforms crenated cells (echinocytes) into cup-shaped ones (stomatocytes), presumably expanding thereby the red cell membrane inner leaflet relative to the outer one (Behn, C., Lübbemeier, A. and Weskamp, P. (1977) Pflügers Arch. 372, 259-268). Whether the Ca-CTC interaction with lipid monolayers may in fact expand the latter, has now been examined by surface tension measurements at the air/water interface. CTC and lipids appeared to compete for the available sites at the air/water interface, contributing additively to its surface pressure. Ca increased both the adsorption rate of the antibiotic to the interface and the CTC-induced surface pressure increment. The latter was not influenced by the subphase pH and ionic strength, or by the type of phospholipid polar head. Correspondingly, CTC-induced cell shape changes should be determined by the pCa values facing either monolayer of the erythrocyte membrane. Both stomatocytes and echinocytes could indeed by obtained with 0.5 mmol . 1(-1) CTC, the cell shape depending on whether the external medium was adjusted respectively to pCa 9 or to pCa 3. Fluorescence microscopy revealed the Ca-CTC complex to be mostly restricted to the cell in stomatocytes and to the external medium in echinocytes. The possibility of inducing alternative cell shapes by varying the transmembrane Ca-CTC distribution, and the demonstration of a Ca-dependent expansion of even relatively compressed lipid monolayers by CTC, together suggest that the Ca-CTC complex may also differentially expand either leaflet of the red cell membrane.

Air↗
PubMed1982

Centruroides toxin, a selective blocker of surface Na+ channels in skeletal muscle: voltage-clamp analysis and biochemical characterization of the receptor.

This paper describes the effects of a toxin from the scorpion Centruroides suffusus suffusus on frog skeletal muscle. The main findings are the following, (i) Centruroides toxin (CssII) blocks the early phase of the inward sodium current in the muscle that arises from influx via Na+ channels in the surface membrane, but it does not affect the late phase of the inward current that represents flux through Na+ channels in the T-tubule membranes, (ii) CssII, in marked contrast to tetrodotoxin, does not affect contraction of the muscle, (iii) Measurements of the binding of 125I-labeled CssII to a partially purified membrane preparation from the muscle indicate that the Kd of the CssII--receptor complex is approximately 0.4 nM. The half-life for the dissociation of this complex is 3 min at 22 degrees C and 16 min at 2 degrees C. Binding of the radiolabeled toxin varies markedly with pH and becomes insignificant at pH greater than 8.5. Proteolytic digestion of the membrane preparation decreases its ability to bind CssII, suggesting that the receptor is a protein. (iv) The number of binding sites for a radiolabeled derivative of tetrodotoxin on the membrane preparation was similar to that for CssII. However, neither tetrodotoxin nor any of seven other neurotoxins and some local anesthetics that alter the functioning of the Na+ channel have any effect on the binding of CssII to the muscle membrane. These results therefore indicate that CssII belongs to a different class of neurotoxins that has a different receptor on the Na+ channel.

Animals↗
PubMed1976

Density and distribution of tetrodotoxin receptors in normal and detubulated frog sartorius muscle.

Tetrodotoxin (TTX) binding was measured in muscles which were either in normal condition or which had been "detubulated" by glycerol-induced osmotic shock. In both cases the binding of TTX was found to saturate at high TTX concentrations. Maximum binding in normal fibers was 35 pmol/g wet weight, and that figure was reduced to 16 pmol/g after glycerol treatment. The dissociation constant for binding to the surface membrane was 3 nM, which is the range of values obtained by electrophysiological measurements of the effect of TTX on the maximum rate of rise of the action potential. The results suggest that the dissociation constant in the transverse tubules may be higher than that in the surface. Control experiments indicate that the effects of glycerol treatment are limited to the accessibility of the receptors to the toxin and result in no alteration of the affinity of the binding site. TTX receptors in the transverse tubules may be recovered after glycerol treatment by homogenization of the fibers. The measurements suggest that the density of sodium channels in surface membrane is about 175/muM2 and that in the transverse tubular membrane is 41-52/mum2.

Action Potentials↗