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Biomedical subjects

G Isenberg

Publications and source records attributed to G Isenberg.

At least 127 records · Page 7Linked to original sources
PubMed1986

Antagonism of forskolin effects by adenosine in isolated hearts and ventricular myocytes.

Adenosine is known to antagonize the effects of catecholamine stimulation in atrial and ventricular tissue; however, its mechanism of action is unknown. Forskolin is an inotropic agent that causes an increase in cyclic AMP (cAMP) levels independent of receptor stimulation. We sought to test whether adenosine could attenuate the effects of forskolin in isolated perfused guinea pig hearts and isolated single ventricular myocytes. In isolated perfused hearts (n = 18), forskolin caused a concentration-dependent increase in left ventricular pressure and dP/dt. Adenosine (5 microM) antagonized the forskolin (0.35 microM)-induced increase in left ventricular pressure and dP/dt by 96 +/- 2 and 92 +/- 4% (means +/- SE), respectively. In contrast, in four hearts, adenosine was ineffective in attenuating the inotropic response to dibutyryl cAMP. In isolated ventricular myocytes (n = 10) 150 nM forskolin caused a significant increase in action potential duration and plateau. In voltage-clamp experiments (n = 8), 150 nM forskolin caused a 39 +/- 3% increase in the calcium current, which was antagonized by adenosine (50 microM) by 80%. Forskolin also caused an increase in contractility, as estimated by sarcomere shortening of the cell. Adenosine, and its analogue N6-R-phenylisopropyladenosine (L-PIA), antagonized the effects of 150 nM forskolin on the action potential and on sarcomere shortening. Dibutyryl cAMP had similar effects as forskolin, but they were not antagonized by adenosine. At higher concentrations of forskolin, above 300 nM, delayed after depolarizations and sustained spontaneous activity occurred that could be abolished by L-PIA. Forskolin caused a concentration-dependent increase in cAMP, measured in isolated ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1985

The role of sodium channels in the effects of the cardiotonic compound DPI 201-106 on contractility and membrane potentials in isolated mammalian heart preparations.

The novel compound DPI 201-106 (4-3-(4-diphenyl-methyl-1-piperazinyl)-2-hydroxypropoxy-1H-indole-carbon itrile) prolonged the action potential duration (APD) and enhanced force of contraction in isolated papillary muscles of the guinea-pig. The effective concentration range was 0.1-3 mumol/l. These effects persisted upon removal of the compound, even after extensive washings. Both prolongation of APD and the positive inotropic effect were readily reversed or prevented after exposure to tetrodotoxin, 3 mumol/l. Slow action potentials of partially depolarized preparations in high potassium solution were hardly influenced by DPI 201-106 (1 mumol/l) or were depressed (3 mumol/l). In isolated myocytes DPI 201-106 induced a slowly decaying net inward current, that disappeared again after exposure to tetrodotoxin. With the exception of the lack of reversibility by washing, these effects were similar to the ones reported previously for the Anemonia sulcata polypeptide ATX II. ATX II and DPI 201-106 did not affect the post-rest contraction. The biphasic response in APD after a transient interruption of stimulation was accentuated by ATX II and became monophasic with DPI 201-106. It is concluded that the effects of DPI 201-106 are also mediated by an interaction with the Na channels, but DPI 201-106 and ATX II probably affect the channels in a different manner.

Animals↗
PubMed1985

Effect of imipramine on calcium and potassium currents in isolated bovine ventricular myocytes.

Isolated bovine ventricular myocytes were investigated with a two-microelectrode voltage clamp technique. The clamp currents were analyzed in terms of ICa and IK. Possible effects on INa were avoided by superfusing the cells with a Na-free medium. Imipramine (IMI) was applied at a concentration of 3.6 microM. Within the initial 3 min (early phase), IMI reduced peak ICa by 38 +/- 9% but IMI did not change the time constants of inactivation, the voltage dependence of peak ICa or its reversal potential. Therefore, we conclude that IMI reduced calcium conductance. After 10 min of exposure (late phase), IMI can also reduce the reversal potential of ICa. The inward rectifying potassium current (IK1) was transiently enhanced by 15 +/- 8% but later (8-10 min) reduced by 19 +/- 4%. Washout of IMI completely reversed all the effects within 10 min. Reduction of ICa diminished the rate of rise and the overshoot of the slow action potential and can explain the shortening of the AP seen in both Na-free and Na-containing media. Possible clinical implications are discussed.

Action Potentials↗
PubMed1985

Monoclonal antibodies localize the exchangeable GTP-binding site in beta- and not alpha-tubulins.

A combination of several methods was used to localize the exchangeable GTP-binding site in the alpha/beta-tubulin heterodimer: direct photoaffinity labeling with [alpha-32P]GTP, specific labeling of alpha- and beta-tubulin by tyrosylation and phosphorylation, respectively, and immunoprecipitation with specific monoclonal antibodies. Direct evidence was obtained that GTP binds exclusively to beta- and not alpha-tubulins.

Animals↗
PubMed1985

The two components in the shortening of unloaded ventricular myocytes: their voltage dependence.

In isolated myocytes from mammalian ventricles a fast and a slow component in the contractile response to depolarizing voltage clamp steps were identified. The potential dependence of the slow component was identical to the activation curve of iCa. The fast component, however, remained at its maximal amplitude at potentials positive to +10 mV (up to +100 mV), in which potential range iCa declined and eventually disappeared. The results suggest that the slow component may be activated by Ca++ entering through sarcolemmal Ca channels, whereas the fast component depends on Ca release from intracellular sites and may depend on both Cai and voltage.

Animals↗
PubMed1985

Extra- and intracellular lanthanum: modified calcium distribution, inward currents and contractility in guinea pig ventricular preparations.

In guinea pig ventricular strips and isolated cells, 0.1 mM LaCl3 blocks contractility and shortens the action potential (AP) in less than 2 min ("early La-effect"). After 30 min, it prolongs the APs which trigger slow contractions ("late La-effect"). These results confirm earlier reports. X-ray microprobe analysis shows that La initially displaces only a small fraction of that Ca which is superficially bound to the sarcolemma. But, since this Ca is completely removed by Ca-free solutions within 2 min, we suggest that La blocks contractility not by displacing superficial Ca but by blocking the Ca inward current iCa. Blocking of iCa is analyzed with voltage clamp experiments. It is not La-specific, and can also be observed with other calcium channel blockers as well. When iCa has been blocked, the membrane can still generate 100-200 ms long plateaus via the sodium inward current iNa. During the late La-effect, the cells internalize La. Intracellular La is detected by x-ray microprobe analysis in cryosections of frozen muscles and as La-precipitates in EM images from freeze substituted preparations. Simultaneously, the cytosol gains Na and Ca, but the plasmalemmal and sarcoplasmic reticulum (SR) membranes are no longer occupied by Ca but by La. The late La-effect on the prolongation of the AP is La-specific. In the absence of extracellular La, it can be induced by pressure injection of La into the cytosol. The long APs are based on an additional La, it can be induced by pressure injection of La into the cytosol. The long APs are based on an additional inward current which is insensitive to Ca-removal, is inactivated by holding potentials of -40 mV, and is TTX-sensitive. We suggest that the current flows through a fraction of original Na-channels that is modified by i.c. La with respect to inactivation and selectivity. We attribute the late re-occurrence of contractility to activator Ca entering from the bath. Ca-entry might be mediated via enhanced Na/Ca-exchange whose rate is increased by the i.c. Na-load. In addition, Ca may enter through the La-modified Na-channels due to their impaired selectivity. Since i.c. La is known to interfere with the Ca-sequestration by the SR, it is expected to impair relaxation.

Action Potentials↗
PubMed1985

Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig).

Cells were isolated by incubating chunks of tissue from the urinary bladder of the guinea-pig in a high potassium, low chloride medium containing 0.2 mM calcium plus the enzymes collagenase and pronase. After isolation, the cells were superfused with a physiological salt solution (PSS) containing 150 mM NaCl, 3.6 mM CaCl2 and 5.4 mM KCl (35 degrees C). Patch electrodes filled with an isotonic KCl-solution were used for whole cell recordings. With a single electrode voltage clamp we measured a capacitance of 50 +/- 5 pF per cell, an input resistance of 200 +/- 25 kOhm X cm2 and a series resistance of 44 +/- 4 Ohm X cm2. The cells had resting potentials of -52 +/- 2 mV. They did not beat spontaneously but responded to stimuli with single action potentials (APs) which rose from the threshold (-38 mV) with a maximal rate of 6.5 +/- 1.8 V/s to an overshoot of 22 +/- 3 mV. The AP lasted for 36 +/- 4 ms (measured between threshold and -40 mV). Continuous cathodal current produced repetitive activity, a pacemaker depolarization followed the AP and preceded the next upstroke. Net membrane currents evoked by clamp steps to positive potentials were composed of an inward and an outward component. The inward component generating the upstroke of the AP was carried by Ca ions (iCa, Klöckner and Isenberg 1985). The repolarization resulted from a potassium outward current iK. Ca-channel blockers (5 mM NiCl2) reduced iK suggesting that (part of) iK was Ca-activated. iK rose within about 100 ms to a peak of 40-200 muA/cm2 from which it inactivated slowly and incompletely. The inactivating iK followed a bell-shaped voltage-dependence, the noninactivating iK an outwardly rectifying one. Both parts had similar steady state inactivation curves with a half maximal inactivation potential at -36 mV and a slope of 9 mV. Repolarization to -50 mV induced outward tail currents which reversed polarity at -85 mV (the calculated potassium equilibrium potential). The amplitude and the time course of the envelope of the tail currents varied in proportion to iK during the prestep. Thus, the tail current is suggested to reflect the turning off of a potassium conductance which had been activated during the prepulse. iK was largely reduced but not blocked by 20 or 150 mM tetraethylammonium (TEA). TEA did not significantly change the resting potential, but it prolonged the AP and facilitated upstroke and overshoot.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1985

Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea pig).

Single smooth muscle cells isolated from the urinary bladder of the guinea-pig were studied at 35 degrees C in a solution composed of 150 mM NaCl, 3.6 mM CaCl2, 1.2 mM MgCl2, 5.4 mM KCl, 20 mM TEA-Cl, 5 mM glucose, 10 mM HEPES/NaOH (pH 7.4). Whole cells were clamped with a single patch electrode. The clamp settled a step from -65 to -5 mV within 260 microseconds, and afterwards the voltage inhomogeneities were less than 2 mV (measured at the cell edge with a second electrode). The calcium inward current iCa was dissected from net currents by blocking potassium outward currents by means of patch electrodes filled with 130 mM CsCl (Klöckner and Isenberg 1985 a). Pyruvate, succinate and oxalacetate in the patch electrode stabilized iCa and prevented its "run down". 140 ms long clamp steps from -65 to -5 mV evoked a net inward current which could be reversibly blocked by 5 mM NiCl2. The "Ni-sensitive" difference current iCa peaked within 2-4 ms to about 1 nA per cell. Afterwards it completely inactivated; the inactivation could be fitted with three exponentials (time constants of 4, 30, and 250 ms, respectively). The half decay time of 16 ms suggests that most of the inactivation resulted from the fast exponential process. The reference current in the presence of Ni was nearly time independent and almost zero; therefore, iCa could be approximated from the net inward current using the zero current as a reference line.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

The electrophysiological properties of the isolated adult heart cell: an overview.

We review the 3 current components iNa, iCa and iK and present those data which have been recently obtained from single cells. We discuss how the new data compare with those from multicellular tissue, and what they contribute to our understanding of the cardiac action potential. For the reasons of space and clarity, some of the problems will be oversimplified and presented incompletely.

Action Potentials↗
PubMed1984

Ca2+-dependent actin-binding phosphoprotein in Physarum polycephalum. Subunit b is a DNase I-binding and F-actin capping protein.

Physarum contains at least two distinct DNase I-binding proteins, i.e. actin and Cap 42 (a + b). The latter, a tight (1:1) complex of Cap 42 (a) and Cap 42 (b) (Maruta, H., Isenberg. G., Schreckenbach, T., Hallmann, R., Risse, G., Schibayama, T., and Hesse, J. (1983) J. Biol. Chem. 258, 10144-10150), is a Ca2+-dependent F-actin capping protein. DNase I binds to Cap 42 (b) but not to Cap 42 (a). Consequently, DNase I-agarose was used for an affinity-purification of Cap 42 (a + b), after its separation from actin by DEAE-cellulose chromatography. Cap 42 (a + b) was dissociated into its subunits when released from DNase I-agarose by 8.8 M formamide. The two subunits were subsequently separated from each other on hydroxylapatite. Both Cap 42 (a) and Cap 42 (b) were Ca2+-dependent F-actin capping proteins that cap the fast growing end of actin filaments and block actin polymerization at this end. Like Cap 42 (a + b), Cap 42 (b) required Ca2+ for its capping activity only when phosphorylated. The phosphorylation of Cap 42 (b) was completely blocked by DNase I or a tertiary complex of Cap 42 (a), actin, and Ca2+. Cap 42 (b) is not identical with native (= polymerizable) actin because (i) Cap 42 (b) was unable to form filaments, (ii) the Cap 42 (b) kinase did not phosphorylate native actin, and (iii) fragmin formed a tight (1:1) complex with native actin but not with Cap 42 (b). Although it is unlikely that Cap 42 (b) is simply a denatured form of actin that has lost its polymerizability during the preparation, it still remains to be clarified whether Cap 42 (b) is a nonpolmerizable actin variant derived from a distinct actin gene or a post-translationally modified form of polymerizable actin.

Actins↗
PubMed1984

Contractility of isolated bovine ventricular myocytes is enhanced by intracellular injection of cardioactive glycosides. Evidence for an intracellular mode of action.

The contractions of isolated bovine left ventricular myocytes were evaluated by optically measuring the extent of unloaded shortening (ES), the maximal rate of shortening (MRS) and the maximal rate of re-lengthening (MRL). Ouabain, digoxin or digitoxin were intracellularly injected by 2 sec long pressure pulses via the microelectrodes. Their i.c. concentration was estimated to be 2-5 nM. Within 1-4 min after the injection, ES, MRS and MRL increased by more than 2-fold. The contractility renormalized within the following 20 min. Injection of solutions without glycosides did not increase the contractility. An interaction of the injected glycoside with the e.c. ouabain receptor could be largely excluded because a) the amount of the released glycoside was too small for e.c. effects, b) 500 nM e.c. antidigoxin, c) 20 mM [K]o or d) covalent binding of digoxin to HSA did not prevent the increase in contractility due to the i.c. injections. Since contractility also increased when the injections were performed at Na-free conditions, [Na]i-load is not necessary for the effect of i.e. glycosides. The increased contractility due to the injected glycosides was not observed when the contractility prior to the injection was already potentiated, e.g. by greater than 3.6 mM [Ca]o or by stimulation at frequencies greater than 1.25 Hz. The results are interpreted by the hypothesis that the i.c. glycosides facilitate the release of activator calcium from the SR. The possible i.c. modes of action are discussed as well as the idea that e.c. applied glycosides internalize and mediate inotropy via the i.e. mechanism.

Animals↗
PubMed1984

Linear electrical properties of isolated cardiac cells.

A frequency domain equivalent circuit analysis of isolated ventricular cells indicated the presence of an internal membrane structure which has a total capacitance four- to sixfold larger than the surface membrane. The internal membrane was mainly attributed to the sarcoplasmic reticulum since other morphological studies have shown that its area is many-fold larger than that of the surface membrane. Corresponding estimates from the transverse tubular system indicate an area less than that of the surface; thus this structure is not a likely candidate for the observed internal capacitance. Measurements in hypertonic solutions showed that the access resistance to the internal membrane reversibly increased as the tonicity was elevated. Freeze-fractured electron microscopic studies confirmed that hypertonic solutions increased the volume of transverse tubular system, which thus appears to have little relation to the access resistance. The most probable source of the access resistance is the diadic junction to the sarcoplasmic reticulum, which therefore would electrically couple it to the surface membrane.

Animals↗
PubMed1984

The effects of the Anemonia sulcata toxin (ATX II) on membrane currents of isolated mammalian myocytes.

The effects of Anemonia sulcata toxin (ATX II) on action potentials and membrane currents were studied in single myocytes isolated from guinea-pig or bovine ventricles. Addition of ATX II (2-20 nM) prolonged the action potential duration without a significant change in resting membrane potential. Concentrations of 40 nM-ATX II or more induced after-depolarizations and triggered automaticity. The effects were reversible after washing or upon addition of 60 microM-tetrodotoxin (TTX). 5 mM-Ni did not modify the effects. The single patch-electrode voltage-clamp technique of Hamill, Marty, Neher, Sakmann & Sigworth (1981) was applied to record membrane currents in response to 8.4 S long depolarizations starting from a holding potential of -90 mV. Currents flowing later than 5 ms after the depolarizing step were analysed. The fast events could not be considered because of insufficient voltage homogeneity. After 2 min of exposure to ATX II (20 nM) the changes in net membrane currents were measured. The difference between the currents in the presence of ATX II and during control was defined as the 'ATX-II-induced current' (iATX). After 4 min of wash iATX disappeared. Within 10 S of exposure to 60 microM-TTX, iATX was blocked completely. At potentials positive to -60 mV, iATX was inwardly directed and decayed slowly but incompletely during the 8.4 S long depolarizing pulse. The rate of decay was faster during clamp pulses to more positive potentials. A high amplitude noise was superimposed on the current trace; its amplitude decreased with more positive potentials. We analysed the voltage dependence of iATX with 'isochronous' current-voltage relations. The 0.1 S isochrone of iATX was characterized by a 'threshold' for negative currents at -60 mV, a branch with a negative slope (k = -7 mV, potential of half-maximal activation (V0.5) = -38 mV, bovine cells) leading to a maximum inward current at -20 mV, and an ascending branch which led to an apparent reversal potential (Erev) around +40 mV. The values measured in guinea-pig myocytes were similar though not identical (k = -5.5 mV, V0.5 = -30 mV, maximum of inward current at -5 mV, Erev = +50 mV). Erev shifted to less positive potentials in later isochrones. Holding the membrane at -45 mV prevented the induction of extra current by ATX II. When the holding potential was then changed to -85 mV, iATX developed within some 2 min. Returning the holding potential to -45 mV blocked iATX with a similar slow time course.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1984

Ionic basis for the antagonism between adenosine and isoproterenol on isolated mammalian ventricular myocytes.

We studied the effects of adenosine and isoproterenol on membrane currents of isolated bovine and guinea pig ventricular myocytes with a two-microelectrode voltage clamp technique. Adenosine (50 microM to 0.2 mM) alone had no effect on any of the membrane currents measured, but it antagonized the effects induced by 10 nM isoproterenol. Peak calcium membrane current was augmented by isoproterenol from a control of 4.8 +/- 0.6 to 8.6 +/- 0.8 nA and adenosine reduced it to 5.7 +/- 0.7 nA (mean +/- SEM of six cells). The inactivation time constant was not altered by isoproterenol alone or isoproterenol plus adenosine, and neither was the voltage dependence of peak calcium membrane current. Thus, the changes caused by isoproterenol could be described as an increase in maximal calcium conductance from 0.86 +/- 0.7 to 1.55 +/- 0.04 mS/cm2 and partially antagonized by adenosine to 0.97 +/- 0.04 mS/cm2. Isoproterenol also increased the non-inactivating component of calcium membrane current from 17 +/- 1 to 24 +/- 4%, and adenosine reduced it to 18 +/- 2% (n = 4). The steady state activation and inactivation variables remained unchanged. Consistent with these effects on calcium membrane current, adenosine completely antagonized the isoproterenol-induced increase of the slow action potentials obtained in sodium-free medium. Isoproterenol increased the steady state outward currents at potentials between -90 and -30 mV (i.e., probable iK1). Adenosine alone had no effect on potassium membrane current, but it antagonized the effects of isoproterenol. Slow action potentials in 25 mM potassium were enhanced by isoproterenol, but were only moderately attenuated by adenosine. Accordingly, in 25 mM potassium the isoproterenol-induced changes in membrane currents were not antagonized by adenosine. This lack of inhibition by adenosine of the isoproterenol effects in 25 mM potassium could not be mimicked by 1-minute-long conditioning prepulses to -45 mV. The results indicate that adenosine by itself (absence of isoproterenol) has no effect on maximal calcium conductance, that the isoproterenol-induced increase in cyclic adenosine 3',5'-monophosphate, which leads to an increase in maximal calcium conductance, is antagonized by adenosine, and that such action can account for the ability of adenosine to attenuate the stimulatory effects of isoproterenol.

Action Potentials↗
PubMed1983

'Cap 90', a 90-kDa Ca2+-dependent F-actin-capping protein from vertebrate brain.

A Ca2+-dependent actin filament-capping protein of 90 kDa was purified from bovine brain using a new and rapid isolation procedure. This basically includes affinity purification on DNase-I agarose. The protein caps the fast-growing end of actin filaments but has no fragmenting or severing activity. Using Triton X-100-extracted cytoskeletons, capping and severing activities of actin-binding proteins become clearly distinguishable from each other.

Actin Depolymerizing Factors↗
PubMed1983

Ca2+-dependent actin-binding phosphoprotein in Physarum polycephalum. I. Ca2+/actin-dependent inhibition of its phosphorylation.

When crude extracts of the slime mold Physarum polycephalum were incubated with ATP and Mg2+ at 35 degrees C, a peptide of approximately 42,000 Da was predominantly phosphorylated. The kinase, separated from the phosphorylatable peptide, phosphorylated neither actin nor fragmin, both proteins of 42,000 Da, the latter known to cap and shorten actin filaments in a Ca2+-dependent manner. The phosphorylatable peptide was phosphorylated only at threonine residue(s), and its phosphorylation was almost completely inhibited by micromolar concentrations of Ca2+ in the extracts. The Ca2+-dependent inhibition of the phosphorylation was reversed by the subsequent addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid but not by trifluoperazine. The Ca2+-dependent inhibition of the phosphorylation required either actin or another, so far unidentified, protein(s) which is distinct from calmodulin. Fragmin reversed the Ca2+/actin-dependent inhibition of the phosphorylation. The Ca2+-dependent actin-binding phosphorylatable protein named Cap 42 (a + b), consisting of two distinct 42,000-Da peptides a and b, was purified to near homogeneity. Peptide b was identified as the phosphorylatable subunit. Substoichiometric amounts of Cap 42 (a + b) reduced the low shear viscosity of F-actin solutions.

Actins↗