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Biomedical subjects

G Isenberg

Publications and source records attributed to G Isenberg.

At least 109 records · Page 6Linked to original sources
PubMed1988

Ca-antagonistic effects of adenosine in guinea pig atrial cells.

In atrial myocytes of the guinea pig, the effects of adenosine (Ado) and acetylcholine (ACh) on Ca currents (ICa) were investigated with the patch-electrode whole cell clamp technique. ICa was dissected from net currents by blocking K currents (IK) with intra- and extracellular Cs ions. ICa was considered as "basal" ICa, since it was not prestimulated by beta-agonists (isoproterenol). T-channel Ca currents were insensitive to Ado or ACh. The antagonism of L-channel Ca currents was maximal with 10 microM Ado or 3 microM ACh, which reduced basal ICa by 35%. From the concentration dependence, a dissociation constant (KD) value of 1.1 microM Ado and a Hill coefficient of -3 were obtained. Ado and ACh were not additive but saturative in reducing basal ICa. Reduction of basal ICa did not modify inactivation time-course, steady-state activation or inactivation, suggesting that Ado reduces the number of functional Ca channels. In myocytes with unblocked IK (KCl electrodes), 3 microM Ado (1 microM ACh) reduced ICa by 30% but increased IK by 300%. It is concluded that the K-agonistic rather than the Ca-antagonistic effect accounts for hyperpolarization as well as for most of the shortening of the action potential and the negative inotropy.

Adenosine↗
PubMed1988

A Dictyostelium mutant with severe defects in alpha-actinin: its characterization using cDNA probes and monoclonal antibodies.

Cells of a Dictyostelium discoideum mutant deficient in binding a monoclonal antibody to alpha-actinin have previously been shown to grow and develop similarly to the wild type and to exert unimpaired chemotaxis as well as patching and capping of membrane proteins. Here we show that the normal 3.0 kb message for alpha-actinin is replaced in the mutant by two RNA species of approximately 3.1 and 2.8 kb. The 3.1 kb RNA was recognized by DNA fragments from all parts of the coding region, while the 2.8 kb RNA hybridized to all but a 3'-terminal fragment. Proteins synthesized in the mutant were analysed using four monoclonal antibodies that in the wild type specifically recognize the 95 x 10(3) Mr polypeptide of alpha-actinin. Cleavage mapping indicated that the binding sites of these antibodies are distributed over a region comprising more than half of the alpha-actinin polypeptide chain. In the mutant, three of the antibodies faintly labelled two polypeptides of 95 x 10(3) Mr and 88 x 10(3) Mr; the fourth antibody, which binds closest to one end of the polypeptide chain, faintly labelled the 95 x 10(3) Mr polypeptide only. The 88 x 10(3) Mr polypeptide most probably lacks the C-terminal portion of alpha-actinin. The binding of an antibody that recognized both polypeptides was quantified by a radio-immuno competition assay using wild-type alpha-actinin as a reference. In a mutant cell extract containing total soluble proteins the antibody binding activity was decreased to 1.1% when compared with wild-type extract. After their partial purification and SDS-polyacrylamide gel electrophoresis the mutant 95 x 10(3) Mr and 88 x 10(3) Mr polypeptides were barely detectable as Coomassie Blue-stained bands, indicating that in the mutant not only certain epitopes of alpha-actinin were altered but the entire molecule is almost completely lacking. When the fitness of mutant cells relative to wild type was determined during growth in nutrient medium, a slight disadvantage for the mutant was indicated, by finding selection coefficients between 0.03 and 0.05.

Actinin↗
PubMed1987

On the interaction of bovine seminal RNase with actin in vitro.

Ribonuclease from bovine seminal plasma (RNase BS) interacts with skeletal muscle actin in the following way: it binds to actin with an apparent binding constant of 9.2 X 10(4) M-1 in 0.1 M KCl, induces the polymerization of actin below the critical concentration in depolymerization buffer, accelerates the salt-induced polymerization of actin even at a molar ratio of RNase to actin lower than 1/100, and bundles F-actin filaments. In the bundles the molar ratio of RNase to actin is about 0.66. Actin inhibits the enzymatic activity of RNase BS. RNase A from bovine pancreas, which is structurally almost identical to the subunits of RNase BS as well as a monomeric form of RNase BS, do not cross-link actin filaments and have a much smaller effect on the polymerization of actin. We conclude that the dimeric structure of the RNase BS, which consists of two identical subunits cross-linked by interchain disulfide bridges, is probably responsible for the bundling activity and the accelerating effect on the polymerization of actin.

Actins↗
PubMed1987

Calmodulin antagonists depress calcium and potassium currents in ventricular and vascular myocytes.

Myocytes isolated from guinea pig ventricles or bovine portal veins were voltage clamped with a single patch electrode. The calmodulin antagonists (CaM-A) calmidazolium, trifluoperazine (TFP), and chlorpromazine acted as Ca antagonists; they reduced the calcium inward current ICa in a voltage- and use-dependent way. For ventricular myocytes, 50% effective concentration (EC50) of calmidazolium was 1 microM, and the EC50 for TFP was 2.5 microM. For vascular myocytes, these numbers were 0.3 and 1 microM, respectively. CaM-A moderately retarded the inactivation time course and shifted the ICa availability curve to more negative potentials. CaM-A were not selective Ca antagonists; other membrane currents such as sodium currents and inwardly and delayed potassium currents were reduced as well (EC50 between 5 and 10 microM). It is unlikely that the above effects require binding of CaM-A to Ca-calmodulin, since reduction of ICa or potassium current (IK) was not modified when 1) the cells were loaded with 100 microM exogenous calmodulin or 2) Ca ions were removed from the extra- and intracellular space. Instead, the unspecific reduction of membrane currents may result from a change in the lipids of the sarcolemma into which CaM-A partition and accumulate.

Animals↗
PubMed1986

Tiapamil reduces the calcium inward current of isolated smooth muscle cells. Dependence on holding potential and pulse frequency.

The calcium currents (ICa) of isolated smooth muscle cells (urinary bladder of the guinea-pig) were analyzed at 35 degrees C and 3.6 mM [Ca]0. The whole cells were voltage-clamped with a single patch electrode which was filled with 150 mM CsCl in order to block potassium outward currents. Tiapamil reduced ICa at concentrations between 1 microM (threshold) and 0.5 mM (complete block). Administration of 10 microM tiapamil at rest reduced ICa by 10% ('initial block'). Repetitive depolarizations (140 ms long pulses to -5 mV, applied at 1 Hz) reduced ICa further in a beat-to-beat fashion. This 'conditioned block' developed with a faster time course and to a greater extent when the stimulation frequency was increased and when the holding potentials were set to more positive levels. Reduction of ICa by tiapamil was facilitated by more positive and attenuated by more negative holding potentials. The influence of holding potential and pulse frequency on the tiapamil effect is discussed in terms of the 'modulated receptor hypothesis'.

Animals↗
PubMed1986

Selection of Dictyostelium mutants defective in cytoskeletal proteins: use of an antibody that binds to the ends of alpha-actinin rods.

A monoclonal antibody, mAb 47-19-2, was used to study the subunit topology of the rod-shaped alpha-actinin molecules of Dictyostelium discoideum and to screen for mutants defective in the production of alpha-actinin. Electron microscopy of rotary-shadowed alpha-actinin-antibody complexes showed binding of mAb 47-19-2 to both ends of the alpha-actinin rods and cleavage of the rods into its subunits, indicating that the two subunits of alpha-actinin extend in an anti-parallel mode through the whole length of the rod. The antibody binding sites were located in close proximity to the sites responsible for actin cross-linking, which is consistent with the blocking activity of the antibody. In a mutant, HG1130, no antibody label was detected in colony blots, and by immunoblotting of mutant proteins separated by SDS-PAGE, only trace amounts of alpha-actinin were found. The mutant showed normal binding of antibodies directed against the actin-binding proteins severin and capping protein. The mutation responsible for the alpha-actinin defect was recessive and located on linkage group I of the genetic map of D. discoideum. HG1130 cells grew on bacteria at a normal rate and also axenically like cells of the parent strain AX2. After starvation the mutant cells expressed the contact site A glycoprotein, a marker of the aggregation-competent stage, and reacted chemotactically to cyclic AMP. The aggregation patterns and fruiting bodies of the mutant appeared to be normal. Patching and capping on the surface of HG1130 cells was induced by antibodies against the contact site A glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinin↗
PubMed1986

Preparation of a toxic derivative of sea anemone toxin II from Anemonia sulcata which has peroxidase activity.

Sea anemone toxin II (ATX II) is a useful tool for investigation of sodium channels in excitable membranes. Coupling of ATX II with microperoxidase, a heme-octapeptide, is described. The isolated cross-linked product possesses both toxic and peroxidase activity. The lowest concentration of the modified toxin that affects the action potential and the membrane currents in single cardiac myocytes is 16 nM. The enzymatically active ATX II derivative may be useful for biochemical applications and as a non-radioactive tracer for electron microscopy studies of excitable tissue.

Action Potentials↗
PubMed1986

Structure of Physarum actin gene locus ardA: a nonpalindromic sequence causes inviability of phage lambda and recA-independent deletions.

Previously we reported that approx. 80% of the genome from the plasmodial slime mold Physarum polycephalum, including all the actin genes, can be cloned only in recBC- sbcB- Escherichia coli hosts [Nader et al., Proc. Natl. Acad. Sci. USA 82 (1985) 2698-2702]. We have now sequenced the actin gene locus ardA. The nucleotide sequence of its coding region is flanked by the typical putative regulatory sequences for transcription initiation and polyadenylation. The coding region is interrupted by five introns, all located at novel positions with regard to those of previously analysed actin genes. Within the ardA gene we have located a 360-bp fragment which comprises exon V and parts of its flanking introns. This region suppresses plaque formation of recombinant lambda phages and causes recA-independent deletions in phages and plasmids. In contrast to our previous hypothesis, this sequence is not a DNA palindrome, but consists of five (dA) X (dT)- and (dG) X (dC)-homopolymers. Both termination of replication and partial unwinding of duplex DNA under torsional stress were detected within the unstable 360-bp region in vitro.

Actins↗
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↗