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G Isenberg

Publications and source records attributed to G Isenberg.

At least 163 records · Page 9Linked to original sources
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↗
PubMed1983

Ca2+-dependent actin-binding phosphoprotein in Physarum polycephalum. II. Ca2+-dependent f-actin-capping activity of subunit a and its regulation by phosphorylation of subunit b.

Cap 42 (a + b), a Ca2+-dependent, actin-binding and phosphorylatable protein consisting of two distinct subunits a and b of 42,000 Da in Physarum polycephalum, has been identified as a new F-actin-capping protein. It capped or bound to the fast growing ends of actin filaments and blocked actin polymerization at this end. The capping activity residing in subunit a and its Ca2+-dependency were regulated by phosphorylation of subunit b; subunit a required Ca2+ for its capping activity when subunit b was phosphorylated, whereas this activity became Ca2+ independent when subunit b was dephosphorylated. Subunit b contained at least two phosphorylatable threonine residues and probably three additional phosphorylation sites. Like cytochalasins and other F-actin-capping proteins, Cap 42 (a + b) was able to induce a rapid depolymerization of actin filaments at the slow growing end, and also to nucleate actin polymerization. However, unlike Physarum fragmin, Cap 42 (a + b) had no severing activity leading to the fragmentation of actin filaments. Our results indicate that Cap 42 (a + b) is the first Ca2+-dependent F-actin-capping phosphoprotein whose phosphorylation regulates its actin-binding and vice versa. A possible mechanism of the capping action of Cap 42 (a + b) in vitro and also its conceivable role in the regulation of the Ca2+/actin-dependent cytoplasmic streaming in plasmodia are discussed.

Actins↗
PubMed1983

12-fold difference between the critical monomer concentrations of the two ends of actin filaments in physiological salt conditions.

We determined the critical monomer concentrations at which association and dissociation reactions are balanced at the two ends of actin filaments. For measurement of the critical concentration of the pointed end, interference with the high dynamics of the barbed end was excluded by capping the barbed ends with an actin filament capping protein isolated from bovine brain. The critical concentration of the pointed end (1.5 microM) was found to be 12- to 15-fold higher than the critical concentration of the barbed end (0.10-0.12 microM) at a temperature of 37 degrees C and physiological salt concentrations (100 mM KCl/1-2 mM MgCl2/0.3 mM EGTA or 0.2 mM CaCl2, pH 7.5).

Actins↗
PubMed1983

Voltage-dependent activation of potassium current in Helix neurones by endogenous cellular calcium.

1. The effect of endogenous Ca on potential-dependent K current IKD, was examined in identifiable neurones of Helix aspersa. The suction pipette method of internal perfusion was used along with a combined voltage-clamp method in which the membrane potential was measured by a separate glass micro-electrode and the current was passed by the suction pipette. Activation of the potential-dependent A current, IA, was prevented by using holding potentials of -40 mV where IA is inactivated and by the addition of the A-current blocker 4-aminopyridine. Activation of K currents by transmembrane Ca current, IKCa, was suppressed by Co substitution for Ca ion extracellularly. 2. Under these conditions, IKD rose to a peak value and then subsided to a steady level. The current-voltage (I-V) relationship for peak IKD had an upward bump at about +50 mV that gave it an S-shape. The I-V curve for steady IKD rose continuously. Peak and steady IKD were reduced by perfusing with EGTA or F ions intracellularly. The EGTA effect occurred at intracellular Ca activity levels below 10(-7) M. Increases in the concentration of EGTAi at constant Cai had no additional effect; however, recovery experiments do not allow us to rule out some direct action of EGTA on IKD. 3. Prolonged extracellular perfusion with Co-substituted solutions also reduced IKD and the effects occurred more quickly when the solutions were made hypertonic or caffeine was added to them. The peak transient was abolished, and the small remaining steady IKD (about 5-10% of normal peak IKD) was blocked by tetraethylammonium. IKD could be restored by the temporary reintroduction of Ca in the extracellular solution. 4. The S-shape of the peak I-V relationship for IKD may be due to Ca released from an endogenous site by membrane depolarization. The reduction of steady and peak IKD to very low values by Ca chelators or prolonged perfusion with Ca-free solutions indicates that Cai is important for activation of these K channels. 5. Three cellular structures were identified in electron micrographs of freeze-fractured neurones that could be involved in potential-dependent endogenous Ca release. These were a restricted extracellularly space, an intracellular membrane system of endoplasmic reticulum that may be fused to the internal face of the plasma membrane (the subsurface cisterns of Henkart & Nelson, 1979), and intracellular vesicles that also may be fused to the plasma membrane.

Animals↗
PubMed1983

Isolated atrial myocytes: adenosine and acetylcholine increase potassium conductance.

Adenosine (Ado), like acetylcholine (ACh), hyperpolarizes and shortens the atrial action potential (AP). To elucidate the underlying mechanism of Ado and ACh actions, intracellular APs and membrane currents were measured in calcium-tolerant single myocytes isolated from guinea pig atria. Both Ado and ACh hyperpolarize the resting membrane to the potassium equilibrium potential (Ek) and cause a marked abbreviation of the AP. Analysis of membrane currents reveal that Ado and ACh increase the steady-state currents. The Ado- and ACh-induced current reverses polarity at -92 mV, a value that corresponds well with a calculated EK of -90 mV. Thus the Ado- and the ACh-sensitive current can be interpreted as a potassium current. It is suggested that Ado, like ACh, increases the potassium conductance via a common mechanism.

Acetylcholine↗
PubMed1983

Actions of adenosine and isoproterenol on isolated mammalian ventricular myocytes.

We investigated the effects of adenosine and isoproterenol on enzymatically dispersed ventricular myocytes from bovine and guinea pig hearts. Intracellular stimulation of relaxed myocytes with regular striation patterns and normal resting potential resulted in action potentials with full plateaus accompanied by contractions. Adenosine in concentrations up to 0.2 mM had no significant effect on any of the action potential parameters or on the basal contractility. In contrast, in the same cells, adenosine effectively antagonized the stimulatory effect of isoproterenol. Isoproterenol (1-10 nM) prolonged the action potentials by 34-41%, displaced the plateau to more positive potentials, and caused a 3-fold increase in the extent of myocyte sarcomere shortening. In the presence of adenosine (5-50 microM), isoproterenol increased the action potential duration by only 8-9%, the shift of the plateau was nearly abolished, and the increase in the extent of myocyte sarcomere shortening was less than 10%. In some of the myocytes, isoproterenol (1-10 nM) induced depolarizing afterpotentials accompanied by aftercontractions. The afterdepolarizations occasionally reached threshold resulting in triggered sustained rhythmic activity. Adenosine (20-50 microM) not only reduced the amplitude of the afterdepolarizations and aftercontractions, but also abolished the sustained rhythmic activity. We conclude, first, that isolated ventricular myocytes respond to isoproterenol and adenosine; second, that adenosine has no direct effect, but effectively antagonizes the stimulatory actions of isoproterenol; third, that findings are consistent with the ones reported for multicellular ventricular preparations; fourth, that adenosine concentrations required to attenuate the actions of isoproterenol are in the range of adenosine concentrations released by cardiac cells when oxygen availability is limited and/or demand is increased; and fifth, that endogenously released adenosine may modulate the electrophysiological and contractile effects of catecholamines.

Action Potentials↗
PubMed1982

Actin filament capping protein from bovine brain.

An actin filament capping protein has been purified from bovine brain. The protein has a native mol. wt. of 63 kilodaltons (kd) with subunits of 36 kd and 31 kd and is globular in shape. It nucleates actin polymerization, inhibits filament elongation and filament interactions, and decreases the steady state viscosity of F-actin in substoichiometric amounts (molar ration 1:1000). In addition, the protein increases the critical concentration for actin polymerization. Neither Ca2+ nor calmodulin affects it action. All these effects can be explained by the binding of the protein to the 'barbed' end of actin filaments leading to a blockade of actin monomer addition at the preferred growing end. This is directly demonstrated by electron microscopy. Concerning the polypeptide composition, Ca2+-independence, mode, and stoichiometry of actin interaction, the protein is similar to the capping protein, previously isolated from Acanthamoeba.

Actin Depolymerizing Factors↗
PubMed1982

Intracellular [Ca2+] transients in voltage clamped cardiac Purkinje fibers.

The Ca2+-activated bioluminescent protein aequorin was used to observe intracellular [Ca2+] transients in voltage clamped canine Purkinje fibers. The pattern of luminescence during a voltage clamp pulse was characterized by two components: L1, which is a rapid initial increase in luminescence and L2, which is a slower, secondary rise of variable configuration. 1. L1, L2, inward current, and contraction were abolished by D 600 (2 microM). 2. Paired clamp pulses. L1 reprimes more rapidly than L2; L1 reprimes within 100 ms, L2 does not. 3. Clamp pulse duration. Peak inward current was the same for 50 ms or 500 ms clamp pulses; L1 was either the same or slightly reduced in 50 ms clamp pulses compared to 500 ms clamp pulses. L2, however, was abolished in repetitively given 50 ms pulses compared compared to repetitively given 500 ms pulses. When 500 ms pulses were alternated with 50 ms pulses, L2 was greater in the 50 ms pulse than in the 500 ms pulse. 4. Clamp pulse potential. In the range-35 to O mV, peak L1 and peak inward current occurred at nearly the same time, had the same threshold potential, and had a similar dependence on membrane potential. In the presence of L2, contractions develop severalfold greater peak tension, time to peak tension is longer, and relaxation is more rapid than in the absence of L2. It is concluded that Ca2+ released from stores accounts for L2 and most of the 'activator calcium'. Ca2+ from another source accounts for L1 and activates a small early component of the contraction. L1 has some properties expected for a signal related to Ca2+ entering via slow inward current, but not via Na/Ca exchange.

Aequorin↗