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Biomedical subjects

G Isenberg

Publications and source records attributed to G Isenberg.

At least 91 records · Page 5Linked to original sources
PubMed1990

Effect of membrane potential on acetylcholine-induced inward current in guinea-pig ileum.

1. The whole-cell patch clamp technique with caesium aspartate internal solution was used with single isolated cells from the longitudinal muscle layer of guinea-pig ileum, to investigate the voltage-dependent gating of ACh-induced inward current. 2. In voltage clamp experiments, at holding potentials ranging from -80 to -30 mV, ACh (300 microM) produced a slow sustained inward current in physiological salt bath solution (PSS). The measurements of the reversal potentials on substituting Na+ by other monovalent and divalent cations showed that this current is through non-selective cation channels (Ins, ACh). 3. During hyperpolarizations, Ins, ACh instantaneously increased in amplitude and then relaxed to a new steady-state level. The I-V relationship of the instantaneous peak was linear with a reversal potential of 0 mV, while that of the steady state was bell-shaped. The time course of relaxation appeared to be monoexponential and its time constants were reduced by stronger hyperpolarizations. 4. These results were not affected by the organic Ca2+ antagonists D600 or nitrendipine (10 microM). Under this condition, maximal chord conductance of Ins, Ach which was observed at 0 mV was about 1.5 nS. The steady-state activation relationship was well fitted by Boltzmann's equation with a half-maximal activation (Vh) of -50 mV and a slope factor (k) of -15 mV at membrane potentials negative to 0 mV, but over 0 mV the degree of activation was again decreased. The time constants for relaxation also appeared to follow a sigmoid curve. 5. In current clamp experiments, superfusion of ACh (300 microM) depolarized the membrane up to -10 to 0 mV. Inward current injection resulting in the moderate hyperpolarization of the membrane (-70 to -80 mV) attenuated ACh-induced depolarization and stronger hyperpolarization (less than -80 mV) abolished it. 6. These results show that ACh-induced depolarization is controlled by the membrane potential, which is explained by the voltage-dependent gating of Ins, Ach.

Acetylcholine↗
PubMed1990

Intracellular calcium ions modulate acetylcholine-induced inward current in guinea-pig ileum.

1. The modulatory effect of internal Ca2+ on the current through the ACh-activated non-selective cation channels (Ins, ACh) was investigated by the whole-cell patch clamp technique in single isolated cells of guinea-pig ileum. 2. Ins, ACh was isolated with caesium aspartate internal solution of low Ca2(+)-buffering capacity (10 microM-EGTA). With preceding depolarizations which evoked voltage-operated Ca2+ currents (ICa), Ins, ACh increased in amplitude and decayed more rapidly. The extent of this 'facilitating' effect depended on the number and duration of the depolarizations. 3. When depolarizing pulses were applied during the sustained phase of Ins, ACh, they were followed by large inward tail currents. These tail currents (tail Ins, ACh) resembled the non-facilitated Ins, ACh recorded without the depolarizing pulse, in regard to voltage-dependent gating and dependence on the extracellular Na+ concentration, thus suggesting that the currents are flowing through the same class of channels. 4. The tail Ins, ACh was apparently composed of two components distinguished by the insensitivity to organic Ca2+ antagonists. The minor component (about 20% of tail Ins, ACh) showed a rapid decay (about 150 ms at -60 mV) which could be attributed to voltage-dependent kinetics. The major component decayed slowly within 5 s and appeared to be related to changes in the intracellular Ca2+ concentration. The latter component was not recorded when Ba2+ or Sr2+ were used as a charge carrier for ICa and was blocked by 10 microM-D600 or nitrendipine, or Cd2+ 0.2-0.5 mM). 5. The tail Ins, ACh increased in proportion to Ca2+ influx when the duration of depolarizing pulses were prolonged from 15 to 200 ms, but this 'facilitating' effect was greatly suppressed when the cell was perfused with 40 mM-EGTA. 6. When the pCa in the pipette was varied using 40 mM-Ca-EGTA, the conductance through Ins, ACh increased in a manner dependent on intracellular Ca2+ concentration. Half-maximal and submaximal activation occurred at about 200 nM and 1 microM, respectively. 7. These results show that the activity of Ins, ACh is very sensitive to the intracellular Ca2+ concentration in the physiological range.

Acetylcholine↗
PubMed1990

Contribution of two types of calcium channels to membrane conductance of single myocytes from guinea-pig coronary artery.

1. Whole-cell and single-channel current recordings were used to study calcium channels in single smooth muscle cells isolated from guinea-pig coronary artery. Potassium currents were blocked by intracellular Cs+ ions. 2. Whole-cell currents were recorded with 10 mM-barium in the bath. Step pulses of 200 ms from a holding potential of -90 mV activated calcium channel current when the depolarization reached -55 to -50 mV. All cells showed a current component which inactivated slowly and incompletely. About half of the cells showed an additional current component with a rapid inactivation time course. Both components were abolished by Cd2+ ions (1 mM) and were reduced by changing the holding membrane potential to -40 mV or by addition of 0.1 mM-Ni2+. 3. Single calcium channel currents were measured in cell-attached patches with 110 or 10 mM-Ba2+ as a current carrier. Two different types of single calcium channel activity were observed. 4. A high-conductance calcium channel was activated near -30 mV with 110 mM-Ba2+ and this threshold was changed to about -60 mV with 10 mM-Ba2+ in the patch pipette. The conductance was 28.0 +/- 1.5 pS (mean +/- S.D.) in 110 mM-Ba2+ and 16.0 +/- 1.0 pS in 10 mM-Ba2+. Dependence of the conductance on the concentration of Ba2+ in the patch pipette followed a Langmuir curve: the apparent dissociation constant of Ba2+ was 8 mM. It was concluded that this channel type corresponds to L-type calcium channels. 5. Another calcium channel was found in these experiments. It had a low conductance and was activated at around -50 mV with 110 mM-Ba2+, and this threshold was shifted to about -70 mV when 10 mM-Ba2+ was the charge carrier. The conductance of this calcium channel was 7.5 +/- 0.6 pS in 110 mM-Ba2+ and 5.5 +/- 1.0 pS in 10 mM-Ba2+. With 10 mM-Ba2+, inactivation of the mean current was slow at potentials -70 to -50 mV, but fast and complete (within 100 ms) at more positive potentials. It was concluded that this type of calcium channel corresponds to T-type calcium channels. 6. With the membrane potential continuously held at -50 to -40 mV (with 10 mM-Ba2+ in the patch pipette), i.e. close to the usual resting potential of these cells, T-type calcium channels were completely inactivated whereas rare openings of L-type calcium channels could be detected.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Acetylcholine activates nonselective cation channels in guinea pig ileum through a G protein.

Acetylcholine (ACh) depolarizes the membrane of mammalian intestinal myocytes by activating a nonselective cation channel (G. D. Benham, T. B. Bolton, and R. J. Lang. Nature Lond. 316: 345-347, 1985; R. Inoue, K. Kitamura, and H. Kuriyama. Pfluegers Arch. 410: 69-74, 1987). Here, we present evidence that occupation of the muscarinic receptor by ACh couples to channel activation via a G protein; the coupling can be blocked by pertussis toxin or by intracellular guanosine 5'-O-(2-thio-diphosphate) (GDP beta S), whereas intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) activates the channel in the absence of ACh. The currents, activated by either ACh or GTP gamma S, are nonadditive, conduct sodium ions, and are similar in their voltage dependence and facilitation by submicromolar calcium ions in the cytosol.

Acetylcholine↗
PubMed1990

Force measurements from voltage-clamped guinea pig ventricular myocytes.

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

Administration, Topical↗
PubMed1990

Isolated guinea pig coronary smooth muscle cells. Acetylcholine induces hyperpolarization due to sarcoplasmic reticulum calcium release activating potassium channels.

Smooth muscle cells, dispersed from the circumflex coronary artery of the guinea pig, were studied with the whole-cell configuration of the patch-clamp. The resting potential of about -40 mV was superimposed by spikelike hyperpolarizations (SLHs) up to -20 mV amplitude. The SLHs resulted from spontaneous transient outward currents (spontaneous TOCs) measured under voltage-clamp (-40 or -50 mV). Acetylcholine (ACh; 10 microM) increased SLHs and TOCs in amplitude and frequency. Atropine blocked the ACh effects. ACh-induced SLHs or TOCs were suppressed by bath application of tetraethylammonium (1 or 10 mM) or by cell dialysis with cesium, suggesting that they result from induction of potassium currents. In cell-attached patches, induction of currents through 130-pS potassium channels was recorded when ACh was bath-applied. An ACh-induced increase in intracellular [Ca2+] is suggested as a second messenger since SHLs and TOCs were suppressed by cell dialysis of 10 mM EGTA. ACh induced SHLs and TOCs in the absence of extracellular calcium. Intracellular application of 5 mg/ml heparin blocked ACh-induced TOCs. When the intracellular calcium stores were depleted by pretreatment with caffeine, the ACh effects were suppressed. Similarly, ACh pretreatment reduced the caffeine-induced outward currents. The results suggested that ACh augments calcium release from the sarcoplasmic reticulum, and the released calcium activates maxi potassium channels. In the single cell, calcium-activated potassium channels generate TOCs and SLHs that sum up to a hyperpolarization of the multicellular tissue.

Acetylcholine↗
PubMed1989

A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.

Actins↗
PubMed1989

The dihydropyridine niguldipine modulates calcium and potassium currents in vascular smooth muscle cells.

1. Vascular smooth muscle cells were isolated from the portal vein and from pial vessels of the cow. They were voltage-clamped with a single patch electrode technique (whole cell recording) in order to analyse the effects of niguldipine on ionic membrane currents. Due to adsorption of niguldipine to plastic and glass, the effective concentrations are lower than the nominal concentrations by a factor of about 3. 2. Niguldipine reduced Ca-currents (ICa of the L-type, voltage operated) at nominal concentrations greater than 0.1 microM up to a complete block at 1 microM (50% block at 0.4 microM). Nominal concentrations between 50 and 200 nM facilitated ICa ('Ca-agonistic effect'). The Ca-agonistic effects of niguldipine showed modest use- but strong voltage-dependence. 3. Niguldipine increased the outward currents at nominal concentrations greater than 10 nM. The extra outward currents reversed at -85 mV, the result suggesting that niguldipine had increased potassium currents, IK. Maximal facilitation of IK by niguldipine was about 400% and was obtained at 1 microM, half-maximal facilitation was obtained with a nominal concentration of 20 nM. 4. Both reduction of ICa and facilitation of IK may contribute to vasodilatation by niguldipine. Due to its greater sensitivity, the effects on IK may dominate.

Animals↗
PubMed1989

X-ray microanalysis of single cardiac myocytes frozen under voltage-clamp conditions.

By means of a patch pipette, an isolated ventricular myocyte was transferred into the taper of a silver holder covered by pioloform film. Once the cell was on the film, the cell was voltage clamped (pulses from -45 to +5 mV at 0.5 Hz). The amount of Ca entry was estimated from the Ca current. When contractility (cell shortening) was potentiated with either five pulses of 0.2 s or four pulses of 1 s, shock freezing was timed 116 or 816 ms after start of the clamp pulse. Electron micrographs from freeze-substituted cells revealed the good preservation of the intracellular compartments. The myocytes were cut at -150 degrees C, and the cryosections were freeze dried. In representative examples, the amount of Ca entry is compared with the subcellular Ca distribution as it is analyzed with energy dispersive X-ray microprobe analysis in cytoplasm, junctional sarcoplasmic reticulum (SR), mitochondria, and the subsarcolemmal space (sarcolemma, peripheral SR, fringe of cytosol).

Animals↗
PubMed1989

Pharmacological modulation of calcium and potassium channels in isolated vascular smooth muscle cells.

Calcium antagonists relax vascular smooth muscle cells (VSM) by decreasing Ca-influx and intracellular Ca-load. In isolated VSM, Ca-influx was measured as Ca-current by the voltage clamp technique applied to a patch of membrane (single-channel current) or to the whole cell (whole-cell current ICa). Gallopamil exerted Ca-antagonism mostly by reducing channel availability, i.e. the probability that the Ca-channel opens upon depolarization. Whole-cell-Ca-currents revealed prominent frequency dependence, i.e. reduction of ICa increased with the number of depolarizations. In addition, the gallopamil effect was voltage-dependent such that depolarized myocytes were more sensitive than hyperpolarized cells. The dihydropyridine nitrendipine abbreviated the life time which the Ca-channel stood in the open state and it hindered the channel to re-open again. Reduction of availability was found only after a prolonged application. In whole cell ICa, nitrendipine accelerated the inactivation time course. The Ca-antagonistic effect was voltage-dependent but not frequency-dependent. Potassium agonists are supposed to activate K-channels thereby hyperpolarizing the membrane, hyperpolarization shuts off the Ca-channels and thereby reduces Ca-influx. The K-agonists cromakalim, (+) niguldipine and diazoxide activated the Ca-dependent maxi K-channel (inside-out patches studied at [Ca2+]c of 50 nmol/l or 500 nmol/l. They increased the open probability mainly by decreasing the long closures between the channel openings. The K-agonists can repolarize the cell once it excited and suppress further excitability.

Animals↗
PubMed1988

The alpha subunit of the GTP binding protein activates muscarinic potassium channels of the atrium.

It has been debated whether the potassium channel of the atrium is activated by the alpha subunit or by the beta gamma subunits of guanine nucleotide binding (G) proteins, which dissociate on activation with guanosine triphosphate (GTP). Therefore, the channel-activating effectiveness of these subunits on isolated guinea pig atrial cells was tested. The activated alpha K subunit from human erythrocytes activated the channel in subpicomolar concentrations. The beta gamma dimer from bovine brain activated the channel in nanomolar concentrations. These results support the view that, physiologically, the alpha subunit activates the channel.

Animals↗
PubMed1988

Effect of reduced Na gradient on electrical activity in isolated bovine and feline ventricular myocytes.

We have studied changes in electrical activity resulting from abrupt alterations of the Na gradient, using ventricular myocytes isolated from feline and bovine hearts. Attempting to investigate the ionic current possibly generated by Na-Ca exchange, we studied the effects of the changes in [Na]o in the presence of 20 mM CsCl to inhibit K currents. To facilitate the effect of Cs, we also used a K-free solution and a patch electrode filled with 150 mM cesium glutamate. The application of 20 mM Nao resulted in hyperpolarization and the action potential duration was reduced. Under voltage clamp, 20 of 45 mM Nao generated an outward current at all membrane potentials investigated. The initial part (100-200 ms) of this current was only partially inhibited by 5 mM NiCl2 which is known to fully block the Ca inward current. However, the outward current generated by the reduced [Na]o was fully inhibited by 20 mM MnCl2 (which presumably inhibits Na-Ca exchange). Our observations extend the work on multicellular cardiac preparations indicating that the outward current elicited by a sudden decrease in Na gradient could be generated by Na-Ca exchange. Although the characteristics of this outward current support certain concepts of the Na-Ca exchange in cardiac muscle, we cannot at present exclude a contribution of other membrane current(s).

Action Potentials↗
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↗