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G Isenberg

Publications and source records attributed to G Isenberg.

At least 91 records · Page 5Linked to original sources
PubMed1993

Stretch-activated nonselective cation channels in urinary bladder myocytes: importance for pacemaker potentials and myogenic response.

Filling of the bladder with urine stretches the myocytes in the wall. Stretch activates nonselective cation channels (SACs) thereby constituting a pacemaking mechanism. Once action potentials are triggered, Ca2+ influx through nifedipine-sensitive Ca2+ channels provides activator Ca2+ for the stretch-induced increase in wall tension (myogenic response). An additional component of myogenic response is independent of nifedipine and membrane potential; Ca2+ influx through SACs is large enough to induce Ca2+ release from intracellular stores.

Action Potentials↗
PubMed1993

The viscoelasticity of entangled actin networks: the influence of defects and modulation by talin and vinculin.

Rheological measurements of the frequency-dependent complex elastic module G*(omega) of entangled F-actin solutions in the frequency range 10(-5)-1 Hz were carried out in three dynamic regimens: 1.) A terminal relaxation from gel-like to liquid-like behaviour measured at frequencies omega < or = tau d-1, 2.) a rubber-type plateau and 3.) a regime determined by chain conformational transitions at frequencies omega > tau i-1. A major point of interest was to clarify whether rheological, high precision measurements can yield quantitative information about the influence of talin and vinculin on the structure, chain dynamics, elasticity and viscoelasticity of actin filaments with time. We show that in the regime reflecting internal chain dynamics (10(-2) to 1 s time domain), F-actin behaves as a random coil of the Rouse type. This contrasts with dynamic light scattering and correlation spectroscopic studies of actin filament flickering, which indicate that filaments behave as semiflexible rods. The internal chain dynamics, which are determined by thermically excited bending undulations, exhibit a persistence length of 0.3-1 microns. Evidence is provided that this discrepancy is due to a cross-over of semiflexible rod behaviour at excitation wavelengths (lambda) below approximately 1 micron to random-coil behaviour at lambda >> 1 micron (expected at a frequency omega approximately 1 Hz). The random coil behaviour is largely determined by defects in actin filaments leading to sharp bends of the chain which act as semiflexible hinges. Talin produces drastic effects on the time course of viscoelasticity during actin polymerization. It promotes the rapid formation of short filament fragments (approximately 1 micron, within time scale of min) which anneal slowly into long filaments (within several hours), most probably by fusion. The viscoelasticity depends on the coexistence of short and very long filaments indicated by the elongation of the rubber plateau. The most dramatic effect is a reduction of the ratio of the terminal (tau d) to the Rouse relaxation time of tau i by more than one order of magnitude (tau d/tau i = 100 compared to ratio tau d/tau i = 2000 for pure actin). From this it is concluded that talin causes a remarkable decrease in the effective segment length of the macromolecule and, thus induces an increase in chain stiffness. Vinculin on the other hand shows no such effect.

Actins↗
PubMed1993

Changes in mitochondrial calcium concentration during the cardiac contraction cycle.

OBJECTIVE: The aim was to examine whether mitochondrial Ca2+ fluxes are high enough to change mitochondrial and cytosolic calcium concentration during the contraction cycle. METHODS: Isolated guinea pig ventricular myocytes were stimulated with paired voltage clamp pulses until contractions were maximal (2 mM [Ca2+]o, 36 degrees C). At defined times of diastole or systole, the cells were shock frozen. Electron-probe microanalysis measured the concentration of total calcium in mitochondria (sigma Ca(mito)) and surrounding cytosol (sigma Cac). Other experiments were performed to evaluate DNP sensitive mitochondrial Ca2+ uptake from depolarisation induced [Ca2+]c transients (K5indo-1 fluorescence). RESULTS: At end of diastole, sigma Ca(mito) was 446 mumol.litre-1. During systole, sigma Ca(mito) increased with a 20 ms delay. A peak sigma Ca(mito) of 1050 mumol.litre-1 was measured 40 ms after start of systole, while 95 ms after start of systole sigma Ca(mito) had fallen to 530 mumol.litre-1. From the changes in sigma Ca(mito) the rates of net mitochondrial Ca2+ flux were estimated at 100 nmol.s-1 x mg-1 protein for Ca2+ influx and 36 nmol.s-1 x mg-1 protein for Ca2+ egress. Decay of sigma Ca(mito) was coupled to a rise in sigma Na(mito). sigma Cl(mito) and sigma K(mito) rose and fell in parallel with sigma Ca(mito), suggesting Ca2+ activation of mitochondrial anion and cation channels. Activation of the non-specific permeability can be excluded. Block of mitochondrial Ca2+ uptake with DNP (100 microM) or FCCP (10 microM) increased the amplitude of the [Ca2+]c transients for 1-3 min by about 50%; evaluation of mitochondrial Ca2+ uptake from DNP sensitive difference signals, however, was hampered by sequestration of mitochondrial Ca2+ into the sarcoplasmic reticulum. CONCLUSIONS: Mitochondrial calcium content changes during each individual contraction cycle; a substantial amount of calcium is taken up during the systole and released during later systole and diastole.

Animals↗
PubMed1993

Ca2+ entry through Na(+)-Ca2+ exchange can trigger Ca2+ release from Ca2+ stores in Na(+)-loaded guinea-pig coronary myocytes.

1. The ionized cytosolic calcium concentration ([Ca2+]i) was monitored in voltage-clamped coronary myocytes at 36 degrees C and 2.5 mM [Ca2+]o using the Ca2+ indicator indo-1. [Ca2+]i was transiently increased by fast application of 10 mM caffeine, and the mechanisms involved in decay of [Ca2+]i were analysed. 2. Resting [Ca2+]i was 166 +/- 62 nM (mean +/- S.D.). Caffeine increased [Ca2+]i within 1-2 s to 1618 +/- 490 nM. In the continuous presence of caffeine [Ca2+]i fell close to resting values with a half-decay time of 5.0 +/- 1.6 s. Wash-out of caffeine induced an undershoot of [Ca2+]i to 105 +/- 30 nM. When caffeine was applied repetitively the [Ca2+]i transients were of reduced amplitude indicating that the store had lost a part of releasable Ca2+. 3. After a 1 s caffeine application [Ca2+]i decayed with a half-time of 2.3 +/- 0.8 s to the undershoot of 112 +/- 57 nM. The decay of [Ca2+]i was largely prevented by 3 mM [La3+]o; after wash-out of La3+ [Ca2+]i fell to the resting value without an undershoot. The results demonstrate that La(3+)-sensitive Ca2+ extrusion contributes to the decay of the [Ca2+]i transient and to the undershoot. 4. With 10 mM [Na+]i, sodium removal from the bath incremented [Ca2+]i in three out of ten cells by 71 +/- 11 nM; in the other cells [Ca2+]i did not change. In the absence of extracellular sodium the decay of [Ca2+]i after wash-out of caffeine was not retarded. 5. To stimulate Na(+)-Ca2+ exchange, cells were dialysed with pipette solution containing 150 mM NaCl. Elevation of [Na+]i had no significant effect on the resting [Ca2+]i (180 +/- 47 nM) or on the caffeine-induced [Ca2+]i transients (peak 1614 +/- 530 nM, half-time of decay 3 s, undershoot 107 +/- 40 nM). 6. With 150 mM [Na+]i, sodium removal resulted in an increase of [Ca2+]i, although responses varied in amplitude (from 130 to 2300 nM) and rate of rise. In the absence of sodium [Ca2+]i remained elevated. After a 1 s caffeine application the undershoot of [Ca2+]i was abolished in sodium-free solution. When caffeine was applied in sodium-free solution, the [Ca2+]i transient decayed to a sustained level and the following caffeine response was attenuated. 7. With 150 mM [Na+]i, the effects of sodium removal were strongly suppressed by a preceding depletion of the Ca2+ stores with caffeine. Ryanodine pretreatment abolished the caffeine-induced [Ca2+]i transients and reduced [Ca2+]i response due to sodium removal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Membrane potential modulates inositol 1,4,5-trisphosphate-mediated Ca2+ transients in guinea-pig coronary myocytes.

1. Vascular smooth muscle cells were isolated from the coronary artery of the guinea-pig. At 2.5 mM [Ca2+]o and 36 degrees C, whole cell membrane currents were recorded under voltage-clamp and the concentration of ionized calcium in the cytoplasm ([Ca2+]i) was monitored by indo-1 fluorescence. 2. At -60 mV, [Ca2+]i was 143 +/- 36 mM (mean +/- S.D.) and was insensitive to clamp steps to +100 mV. During 1 min application of acetylcholine (ACh, 10 microM) [Ca2+]i increased within approximately 2 s to 1480 +/- 250 nM. During the subsequent slow decay, [Ca2+]i was transiently increased by depolarizing clamp steps and decreased during hyperpolarizing steps. [Ca2+]i transients in response to caffeine (10 mM) could not be modulated by voltage steps. The results suggest that modulation of [Ca2+]i by membrane potential involves inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release (IICR). 3. Modulation of IICR by membrane potential did not depend on sarcolemmal Ca2+ fluxes; it persisted after block of sarcolemmal Ca2+ fluxes with 3 mM lanthanum or after a change to nominally Ca(2+)-free bathing solutions. 4. Modulation of [Ca2+]i by membrane potential was recorded during cell dialysis of 50 microM GTP-gamma-S in the absence of ACh. Cell dialysis of exogenous Ins(1,4,5)P3 (50 or 100 microM) did not mimic the effects. The sensitivity of [Ca2+]i to depolarizing clamp steps was also induced by cell dialysis of lithium ions which, presumably, inhibited the breakdown of Ins(1,4,5)P3. The results are compatible with the idea that the membrane potential modulates the liberation of Ins(1,4,5)P3. 5. Modulation of IICR by membrane potential is discussed as a new mechanism that contributes to the regulation of activator calcium and to the modulation of contraction in vascular smooth muscle cells.

Acetylcholine↗
PubMed1993

Microheterogeneity of subsarcolemmal sodium gradients. Electron probe microanalysis in guinea-pig ventricular myocytes.

1. The effect of stimulation on possible subsarcolemmal sodium accumulation was studied in ventricular myocytes (2 mM [Ca2+]o, 36 degrees C). By trains of eighteen paired voltage-clamp pulses (180 ms to 0 mV, 20 ms to -45 mV, 180 ms to +50 mV, 620 ms to -45 mV) unloaded contractions were potentiated to an optimum. 2. Potentiation reversibly enlarged and prolonged the diastolic tail currents due to Na(+)-Ca2+ exchange. Eighteen pulse pairs were estimated to provide a sodium influx that could increment the total intracellular sodium concentration (sigma Na(i)) by no more than 0.5 mM. 3. Potentiation reversibly increased the current at +50 mV and made it more noisy. Cell-attached recordings with a second electrode attributed this noise to the activation of K+ (Na) channels. In inside-out patches, a comparable channel activity was obtained with 40 mM sodium. Hence, the cell-attached recordings suggest that potentiation can increase intracellular sodium concentration to 40 mM. 4. Electron probe microanalysis (EPMA) measured sigma Na in a volume within 20 nm of the inner side of the sarcolemma. Potentiation reversibly increased sigma Na20nm to 40 +/- 7 mM. When stimulation was terminated, sigma Na20nm fell within 8 s to 37 +/- 8 mM and within 3 min to 19 +/- 6 mM. In unstimulated cells sigma Na20nm was 17 +/- 5 mM. 5. In potentiated cells, shock-frozen at early systole, sigma Na fell with a space constant of 28 nm from the sarcolemma to the centre; at 1 microns distance sigma Na was 12 +/- 3 mM. The steep gradient suggests that sodium does not freely diffuse and sigma Na20nm is controlled by transmembrane fluxes rather than by cell dialysis. 6. sigma Na20nm data were distributed with peaks at 5, 30 and 60 mM. Quantitative elemental digital imaging demonstrated patches with 60-80 mM sigma Na20nm alternating with others of 0-15 mM sigma Na20nm. This 'sodium microheterogeneity' suggests that Ca2+ efflux at low sigma Na20nm and K+(Na) channel activation at high sigma Na20nm can operate simultaneously.

Animals↗
PubMed1993

Properties of stretch-activated channels in myocytes from the guinea-pig urinary bladder.

1. Stretch-activated channels (SACs) were analysed on patches attached to myocytes isolated from the guinea-pig urinary bladder. At 22 degrees C application of -2 to -4 kPa to the patch electrode induced SACs at a density of one to two per patch (3-5 M omega electrodes). 2. With electrodes containing 145 mM K+, 20 mM TEA and 2 mM Mg2+, the single channel current followed a linear I-V curve with a slope conductance of 39 +/- 5 pS (mean +/- S.D.) and a reversal potential of 2 +/- 6 mV. Substitution of chloride by aspartate ions left both parameters unchanged suggesting that the anions do not contribute to the currents. 3. Hyperpolarization from -30 to -80 mV did not open channels by itself but increased channel activity (NPo; where N is the number of channels in the patch and Po is the probability of the channel being open) twofold. The hyperpolarization-induced increase in NPo can be attributed to a reduction of long closures. At positive patch potentials numerous blank records strongly diminished NPo. 4. Inward currents through SACs can be carried by a variety of cations. In the presence of 2 mM Mg2+, the respective channel conductance was 40 +/- 4 pS for 140 mM K+ > 34 +/- 2 pS for 140 mM Na+ > or = 33 +/- 6 pS for 140 mM Cs+ > 19 +/- 2 pS for 110 mM Ba2+ > 17 +/- 2 pS for 110 mM Ca2+. 5. Reduction of CaCl2 from 110 to 10 mM did not change the conductance but shifted the reversal potential from +7 to -7 mV; the reversal potentials suggest that SACs are slightly more permeable for Ca2+ than for K+. 6. In the absence of divalent cations, the conductance of K+ was 82 +/- 4 pS for inward but 45 pS for outward currents. Addition of either 2 mM Ca2+ or 2 mM Mg2+ reduced the conductance for inward currents to 40 pS. 7. The change from 140 to 14 mM KCl plus 136 mM Tris-Cl reduced the conductance from 82 to 56 pS whereas the reversal potential shifted only from -4 to -9 mV. When 20 mM K+ and 300 mM sucrose were applied, the conductance fell to 39 pS and the reversal potential shifted by -30 mV. The results suggest that Tris+ can permeate through SACs when extracellular divalent cations are absent.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Talin anchors and nucleates actin filaments at lipid membranes. A direct demonstration.

Platelet talin nucleates actin assembly as we show here directly by using rhodamine-phalloidin labelling of actin filaments. Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein-lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of actin filament growth at lipid interfaces have been visualized.

Actin Cytoskeleton↗
PubMed1992

Probing actin and liposome interaction of talin and talin-vinculin complexes: a kinetic, thermodynamic and lipid labeling study.

Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) talin-nucleated actin polymerization and (b) insertion of talin into lipid bilayers. Calorimetric data show ternary complex formation between talin, vinculin, and actin. Actin-talin, actin-vinculin and actin-(talin-vinculin) binding and rate constants as well as actin polymerization rates for all three protein species have been determined by steady state titration, stopped-flow, and fluorescence assay. In contrast to an increase of the polymerization rate by a factor of less than 2 for actin-talin and actin-(talin-vinculin) when lowering the temperature, we measured a decrease in rates for actin alone and actin-vinculin. The overall equilibrium constants (Keq) in the van't Hoff plot proved linear and were of one-step reactions. Thermodynamic data exhibited signs of van der Waal's binding forces. Using the photoactivatable lipid analogue [3H]PTPC/11, which selectively labels membrane-embedded hydrophobic domains of proteins, we also show that talin partially inserts into the hydrophobic bilayer of liposomes. This insertion occurs in a similar manner irrespective of preincubation with vinculin.

Actins↗
PubMed1992

Potentiation of contraction as related to changes in free and total intracellular calcium.

In voltage-clamped guinea-pig ventricular myocytes, we studied the potentiation of contraction in dependence on the concentration of intracellular calcium; ionized calcium [Ca2+]c was measured by Indo-1 microfluospectroscopy and total calcium (sigma Ca) by electronprobe microanalysis (EPMA). After a 15 min rest period, [Ca2+]c was approx. 90 nM and sigma Ca was below the detection limit (80 microM) in myoplasm (sigma Ca(myo)), junctional sarcoplasmic reticulum (sigma CaSR) and mitochondria (sigma Ca(Mito)). Post rest, repetitive clamp steps (1 Hz) potentiated extent and rate of shortening by 300%. In the literature, post-rest potentiation is attributed to the replenishment of SR with releasable calcium; by EPMA the postulated increase in sigma CaSR was measured directly. Post-rest, the peaks of systolic [Ca2+]c transients increased, however only by 40%. In addition, a moderate increase of end-diastolic [Ca2+]c was measured. In an other series of experiments, contraction was potentiated by 800% increase by means of paired voltage-clamp pulses (1 Hz, 36 degrees C, 2 mM [Ca2+]o). In the potentiated state, end-diastolic [Ca2+]c was 180 nM and sigma Ca(myo) was 0.65 mM. During systole, [Ca2+]c peaked within 20 ms to 950 nM. sigma Ca(myo) rose within 20 ms to 1.4 mM and fell within 40 ms to 1.1 and within 90 ms to 0.8 mM. In contrast, the time course of contraction was slow and peaked at a time (130 ms) when the [Ca2+]c and sigma Ca(myo) transients were finished. We suggest that Ca2+ bound to troponin C (TnC) controls only the onset but not the time course of myofilament interaction. From [Ca2+]c and sigma Ca(myo) we estimated a Ca2+ buffering capacitance of 1.5 mmol sigma Ca(myo) per pCa change, only a fraction of which can be attributed to Ca2+ binding sites on TnC. A model explaining the results requires the assumption of 0.6 mM additional slow, high affinity Ca2+ sites and 2 mM fast, low affinity Ca2+ sites. We discuss that end-diastolic Ca2+ binding to these sites contributes to the potentiation of contraction. Junctional SR. At the end of diastole sigma CaSR was 2.4 mM which is 4 times larger than sigma Ca(myo). This difference disappeared 20 ms after depolarization (sigma CaSR 1.1 mM), within another 20 ms it largely recovered (sigma CaSR 2.0 mM). These properties suggest that the junctional SR is a compartment suitable not only for Ca2+ release but also for rapid Ca2+ reuptake. Mitochondria. Paired-pulse potentiation increased end-diastolic sigma Ca(Mito) significantly (0.4 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Ca2+ influx through voltage- and purinoceptor-operated channels estimated from [Ca2+]c signals (myocytes from guinea-pig urinary bladder).

The rise in free cytosolic calcium was studied by a combination of the techniques of microspectrofluorometry and whole-cell patch clamp. By comparing the membrane currents with their effect on [Ca2+]c, the relative importance of Ca2+ influx could be quantified for both L-type Ca2+ channels and non-selective channels activated by extracellular ATP.

Adenosine Triphosphate↗
PubMed1992

Arginine-vasopressin induces mode-2 gating in L-type Ca2+ channels (smooth muscle cells of the urinary bladder of the guinea-pig).

The effect of arginine-vasopressin (AVP, 0.1 microM) on elementary Ca2+ channel currents (L-type) was studied in cell-attached patches with 10 mM BaCl2 as the charge carrier. At a constant potential of -30 mV, bath applied AVP increased the channel openness (NPo) by a factor of 4.7 +/- 3.0 (mean +/- SD, n = 9), the effect resulted from an increase in the frequency of opening (factor 2.5 +/- 0.8) and from a longer mean open time. Under control, openings longer than 5 ms contributed only 4% of the total, however, with the application of AVP this contribution increased to 29%. Under control, the open times were distributed along a single exponential (tau o1 = 0.8 +/- 0.4 ms), a double exponential distribution was obtained during AVP (tau o1 = 0.8 +/- 0.5 ms, tau o2 = 7.5 +/- 0.7 ms). The Ca2+ agonist BAYk8644 (1 microM) changed the open time distribution similarly to AVP (tau o1 = 1.0 +/- 0.5 ms, tau o2 = 9 +/- 2.8 ms). With 1 microM BAYk8644 in the bath, AVP did not significantly increase the relative contribution of long openings, however, AVP increased the frequency of openings by a factor of 2.0 +/- 1 (n = 6). The results are compatible with the idea that AVP can change the gating of L-type Ca2+ channels from mode 1 to mode 2.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

ATP suppresses activity of Ca(2+)-activated K+ channels by Ca2+ chelation.

Ca(2+)-activated maxi K+ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pfügers Arch 417:517, 1990), channel activity (NPo) was stimulated by 3 microM [Ca2+]c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na2ATP. The following results suggest that suppression of NPo by Na2ATP is due to Ca2+ chelation and hence reduction of [Ca2+]c and reduced Ca2+ activation of the channel. The effect was absent when Mg ATP was used instead of Na2ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca2+]c was buffered with 10 mM HDTA (apparent pKCa 5.58) instead of EGTA (pKCa 6.8). A Ca(2+)-sensitive electrode system indicated that 1 mM Na2ATP reduced [Ca2+]c in 1 mM Ca-EGTA from 3 microM to 1.4 microM. Na2ATP, Na2GTP, Li4AMP-PNP and NaADP reduced measured [Ca2+]c in parallel with their suppression of NPo. After the Na2ATP-induced reduction of [Ca2+]c was re-adjusted by adding either CaCl2 or MgCl2, the effect of Na2ATP on NPo disappeared. In vivo, intracellular [Mg2+] exceeds free [ATP4-], hence ATP modulation of maxi K+ channels due to Ca2+ chelation is without biological relevance.

Adenosine Triphosphate↗