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Biomedical subjects

R Rüdel

Publications and source records attributed to R Rüdel.

At least 37 records · Page 2Linked to original sources
PubMed1999

Malignant hyperthermia causing Gly2435Arg mutation of the ryanodine receptor facilitates ryanodine-induced calcium release in myotubes.

We have investigated if cultivated muscle cells from malignant hyperthermia (MH) patients can be distinguished pharmacologically from controls. Muscle specimens from four individuals carrying the Gly2435Arg mutation of the skeletal muscle ryanodine receptor protein (RYR1) and from four controls were used to culture myotubes. Resting intracellular calcium concentration ([Ca2+]i) of MH myotubes was similar to controls. However, when ryanodine 0.5 mumol litre-1 was added, the kinetics of the increase in the calcium signals in MH and control cells were significantly different; the time for half maximum increase was mean 197 (SD 131) s for MH cells and 474 (61) s for controls (n = 80 cells each). On average, the area under the MH response curves was twice the control value. These results give rise to hopes that the phenotype of MH can be characterized using cultured human muscle and that a culture-based test for MH susceptibility may eventually be developed.

Adolescent↗
PubMed1999

Mutant channels contribute <50% to Na+ current in paramyotonia congenita muscle.

An important question in the pathophysiology of dominantly inherited diseases, such as channelopathies, is the level of expression of the mutant protein. In our study, we address this issue by comparing the gating defects of two human muscle Na+ channel mutants (R1448C and R1448P) causing paramyotonia congenita in native muscle specimens from two patients with those of the same mutant recombinant channels expressed in human embryonic kidney (HEK-293) cells. Patch-clamp recordings of transfected HEK-293 cells revealed a pronounced slowing of the Na+ current decay, a left-shifted and decreased voltage dependence of steady-state inactivation, and an increased frequency of channel reopenings for mutant compared with wild-type channels. For R1448P channels, inactivation was almost six-fold and for R1448C it was three-fold slower than for wild-type channels. The same defects, though less pronounced, as expected for a disorder with dominant inheritance, were observed for muscle specimens from paramyotonia congenita patients carrying these mutations. Quantitative kinetic analysis of Na+ channel inactivation in the paramyotonic muscle specimens separating wild-type from mutant channels suggested that no more than 38% of the channels in the paramyotonia congenita muscle specimen were of the mutant type. Our data raise the possibility that variability in the ratio of mutant to wild-type Na+ channels in the muscle membrane has an impact on the clinical severity of the phenotype.

Amino Acid Substitution↗
PubMed1998

The dominant chloride channel mutant G200R causing fluctuating myotonia: clinical findings, electrophysiology, and channel pathology.

Clinical, electrophysiological, and molecular findings are reported for a family with dominant myotonia congenita in which all affected members have experienced long-term fluctuations of the symptom of myotonia. In some patients myotonia is combined with myalgia. The myotonia-causing mutation in this family is in the gene encoding the muscular chloride channel, hCIC-1, predicting the amino acid exchange G200R. We have constructed recombinant DNA vectors for expression of the mutant protein in tsA201 cells and investigation of the properties of the mutant channel. The most prominent alteration was a +100-mV shift of the midpoint of the activation curve. Therefore, within the physiological range the open probability of the mutant channel is markedly smaller than in wild-type. This shift is likely to be responsible for the myotonia in the patients. The fluctuating symptoms of this chloride channelopathy are discussed with respect to short-term fluctuations of myotonia in the sodium channelopathy of potassium-aggravated myotonia.

Adolescent↗
PubMed1998

Transient weakness and compound muscle action potential decrement in myotonia congenita.

Twenty-five Turkish patients with recessive myotonia congenita (RMC), 16 of whom had genetic confirmation, were studied. Nineteen had transient weakness. In the upper extremities, onset age of transient weakness was usually in the early teens. All untreated RMC patients had a compound muscle action potential decrement of > or =25%, usually above 50%, with repetitive nerve stimulation at 10/s for 5 s. Patients with other nondystrophic diseases with myotonia, except 1 patient with dominant myotonia congenita, had no transient weakness and a CMAP decrement below 25%.

Action Potentials↗
PubMed1998

Myotonic ADR-MDX mutant mice show less severe muscular dystrophy than MDX mice.

In Duchenne muscular dystrophy (DMD) and its murine model, the dystrophic mouse (MDX), the skeletal musculature lacks dystrophin. The presumed function of this cytoskeletal protein is to protect the sarcolemma against mechanical stress during muscle activity. To test this hypothesis in vivo, we bred a double mutant mouse that combines two genetic defects: the dystrophin-deficiency of the MDX mouse and the Cl- channel myotonia of the arrested development of righting response (ADR) mouse. We hypothesized that high mechanical muscle activity would aggravate muscular dystrophy in double mutant ADR-MDX mice. On the contrary, ADR-MDX mice showed fewer signs of muscle fiber necrosis and fibrosis than MDX mice at all ages. Plasma creatine kinase levels were slightly increased in ADR-MDX, but significantly lower when compared to MDX mice. Sections of ADR-MDX muscle showed a uniform pattern of oxidative muscle fibers. Similar findings have been obtained in dystrophin-positive ADR mice, they result from a complete fiber-type IIB to IIA transformation in myotonic muscle. Our results suggest that small, oxidative fibers of myotonic mice are less sensitive to dystrophin deficiency. Therefore, ADR-MDX mice develop less severe muscular dystrophy than MDX mice do, although their muscles are continually stressed. The new ADR-MDX double mutant mouse is the first animal model combining both a dystrophinopathy and a channelopathy. The results presented here give new insights into the pathomechanism of muscular dystrophy and may be helpful for the therapeutic management of DMD.

Age Factors↗
PubMed1997

Antisense oligonucleotides discriminating between two muscular Na+ channel isoforms.

Various 15-mer antisense oligodeoxynucleotides (aODNs) were constructed against RNAs coding for two closely related isoforms of the voltage-dependent Na+ channel, i.e. those of human heart (hH1) and skeletal (hSkM1) muscle. When translated in vitro, either RNA yielded a 220 kDa band on polyacrylamide gels, indicating that the translation product had full length. Of six different aODN constructs developed against hH1 RNA, two each inhibited translation completely, moderately or not at all, depending on the target position. The specificity of the effect (no cross reaction at 10 microM) was confirmed by incubation with 15-mer aODNs against hSkM1 RNA. The most effective aODNs were those hybridizing between bases 3840 and 3880 of hSkM1 RNA and the homologous segment of hH1 RNA. When either of the RNAs was co-injected with its most effective (phospho rothioate-capped) aODN into Xenopus oocytes, the production of Na+ channels was strongly suppressed (relative INa for hSkM1: 0.08 +/- 0.05 times control, n = 14; for hH1: 0.11 +/- 0.08, n = 11). We conclude that aODNs are able to discriminate between closely related RNAs. The efficacy of an aODN depends strongly on its RNA target position.

Animals↗
PubMed1997

Probing the major skeletal muscle chloride channel with Zn2+ and other sulfhydryl-reactive compounds.

The sensitivity of the human skeletal muscle Cl- channel, hClC-1, towards various sulfhydryl-reactive agents was tested with the channel expressed in Xenopus oocytes and in human embryonic kidney cells. External but not internal Zn2+, at 1 mM, substantially reduced the current without affecting activation parameters. External Cd2+ and Hg2+ as well as organic mercurial compounds reduced the Cl- currents to a similar degree. With the mutant channel hClC-1 D136G, presumed to have a defective voltage sensor, external Zn2+ also reduced the current without effect on the altered gating. These findings suggest that hClC-1 contains cysteine residues near the extracellular face that may directly influence ion conduction. Since Zn2+ can also bind to histidine side chains, we tested the effect of compounds with either more cysteine- or more histidine-specificity. The results confirm the involvement of cysteine(s) in the observed effects but do not exclude the involvement of histidine(s).

Chloride Channels↗
PubMed1997

Intracellular calcium chelator BAPTA protects cells against toxic calcium overload but also alters physiological calcium responses.

The effect of the membrane-permeant calcium chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM) on ionomycin-induced cellular calcium overload was studied in single differentiated NH15-CA2 neuroblastoma x glioma hybrid cells. To monitor [Ca2+]i we used the fluorescent indicator Fura-2. Preincubation of the cells with 3 microM BAPTA/AM reduced the number of cells showing deregulation of [Ca2+]i during ionomycin-induced calcium influx. The calcium transients elicited by application of KCl were also severely affected by the chelator. These transients, although varying from cell to cell in shape, amplitude and duration, are well reproducible in individual cells. After incubation of cells for 1 h with 0.3-30 microM BAPTA/AM the time course of these cellular transients was markedly slowed. At 1 microM BAPTA/AM, the time constant of decline of [Ca2+]i was increased by a factor of 4.1 +/- 2.4 (n = 14) and the amplitude was reduced to about 50%. With 30 microM BAPTA/AM, the K(+)-induced calcium transients were almost completely inhibited. We conclude that intracellularly loaded calcium chelators may be used for the prevention of [Ca2+]i-induced cell damage, however, at the expense of a disturbed calcium signalling.

Animals↗
PubMed1996

The human endogenous local anesthetic-like factor (ELLF) is functionally neutralized by serum albumin.

The cerebrospinal fluid (CSF) of patients with multiple sclerosis or Guillain-Barré syndrome contains a factor that inhibits excitation of nerve and muscle cells like local anesthetics. CSF samples containing the endogenous local anesthetic-like factor (ELLF) were analyzed by gel filtration chromatography and ultraviolet (UV) absorption at 210 nm. The active component was in a single peak corresponding to a molecular weight of 600-800 Da. This peak was decreased and the Na+ channel blocking activity was neutralized by the addition of 40 g/l human serum albumin to the CSF. When the albumin was separated from the CSF/albumin mixture by acetonitrile treatment, the Na+ channel blocking activity reappeared. The ELLF and its neutralization may be of relevance for the clinical fluctuations known with these diseases.

Cerebrospinal Fluid↗
PubMed1996

On the nature of endogenous antiexcitatory factors in the cerebrospinal fluid of patients with demyelinating neurological disease.

The cerebrospinal fluid (CSF) of patients with demyelinating neurological disease, such as Guillain-Barré syndrome or multiple sclerosis, contains factors that inhibit the excitatory Na+ current. Such antiexcitatory factors are occasionally also detectable in CSF from patients with other neurological diseases but were absent from an artificial CSF containing all major CSF constituents (electrolytes, amino acids, vitamins, metabolites, albumin). In an attempt to characterize these factors, unphysiological pCa or pH values were excluded by the application of the Ca2+ chelator EGTA and the use of buffers. Heating the CSF for 10 min to 95 degrees C or digesting it with proteases did not destroy the antiexcitatory potency. Fractionation of the CSF contents according to molecular weight showed that the factors have a molecular weight < 3 kD. This excludes proteins, such as antibodies or cytokines, as candidates. Small peptides are known to be resistant to some proteases and heating.

Adolescent↗
PubMed1996

Mechanism of voltage-dependent gating in skeletal muscle chloride channels.

Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels.

Animals↗
PubMed1996

Ether lipid-induced cell damage of neuroblastoma cells is only weakly correlated with increased intracellular Ca2+ levels.

The cell-damaging action of the ether lipid ET-18-OCH3 was studied in single NH15-CA2 neuroblastoma x glioma hybrid cells using light microscopy, and correlated with changes of the free intracellular calcium concentration, [Ca2+]i, as measured with the fluorescent Ca2+ indicator Fura-2. Addition of 3-100 microM ET-18-OCH3 to the cultures caused disintegration of neurites, cell rounding and detachment of the cells from the bottom of the culture dish. The effects occurred within 30-240 min, the faster, the higher the ET-18-OCH3 concentration. Also [Ca2+]i increased in a concentration-dependent manner, however, within seconds, and stayed high during the recording time of 20 min. The presence of 50 microM lanthanum or gadolinium ions prevented the ET-18-OCH3-induced increases of [Ca2+]i, but had no effect on neurite destruction and cell rounding. Preincubation with 1 mM diisopropylfluorphosphate or 100 microM leupeptin, both membrane-permeant inhibitors of intracellular proteases, did not prevent the effects. Nor was neurite destruction prohibited in the presence of 10 microM of the actin-stabilizing agent phalloidin or 2 microM taxol, a microtubule-stabilizer. We conclude that [Ca2+]i, although being increased during ET-18-OCH3-induced cell damage, is not the key factor of cell destruction.

Animals↗
PubMed1996

Channelopathies: the nondystrophic myotonias and periodic paralyses.

The term channelopathy does not indicate a new group of neuromuscular conditions, but a re-orientation of well- and long-known muscular conditions, the congenital myotonias, and the periodic paralyses. Although, in the past, they have overlapped clinically here and there, both groups were classified differently, as myotonias and as metabolic myopathies, respectively. The discovery of mutations in several ion channels has rewritten nosography of these disorders and procured a new term, the channelopathy-clinical, electrophysiological, and molecular genetic details of which are discussed in this chapter.

Adolescent↗
PubMed1995

Human serum albumin enhances sodium currents in NH15-CA2 neuroblastoma x glioma hybrid cells.

The effect of human albumin on neuronal voltage-dependent Na+ channels was studied in NH15-CA2 neuroblastoma x glioma hybrid cells. At an albumin concentration of 32 g/l, the transient Na+ currents, elicited at 1 Hz and recorded with the whole-cell technique, were increased to 125% the control value. The effect was complete within 2-4 s of extracellular albumin application; 35 s after washout of the protein it was not yet fully abolished. The albumin effect is mediated by a shift of the voltage dependence of steady-state inactivation of the Na+ channels towards more positive potentials. The activation of the channels is not noticeably influenced.

Albumins↗
PubMed1995

Chloride currents across the membrane of mammalian skeletal muscle fibres.

1. Chloride currents through the membrane of rat psoas muscle fibre segments were investigated with a double Vaseline gap under conditions minimizing the currents of other ion species. 2. In Cl(-)-free solutions a time- and voltage-independent conductance of 1.1 +/- 0.4 microS was observed. 3. As with intact fibres, the steady-state Cl- conductance was 2.5 +/- 0.9 mS cm-2; the halide selectivity was Cl- > Br- > I-, and Cl- currents were completely blocked by 0.1 mM 9-anthracene carboxylic acid (9-AC). 4. Voltage steps from -85 mV to between -125 and +55 mV elicited currents with deactivation upon hyperpolarization and activation upon depolarization. Activation was fitted with two exponentials, the smaller time constant increasing from 37.5 ms at +55 mV to 67.0 ms at -5 mV, the larger time constant (450 ms) being independent of potentials more positive than -5 mV. The two deactivation time constants ranged between 30.6 (-105 mV) and 99.3 ms (-35 mV), and 139.4 (-105 mV) and 738.5 ms (-35 mV). 5. The activation curve was fitted with a Boltzmann distribution (half-maximum, -39 mV; slope at inflexion point, 1/17.2 mV). Deactivation was incomplete. At very negative potentials about one-quarter of the maximum number of channels were open. 6. When tested with 5 and 61 mM intracellular Cl- concentration ([Cl-]i) the kinetic parameters were not different. 7. During depolarizations lasting > 5 s, activation was followed by a decline. With progressively longer prepulses going positive to the reversal potential and test pulses going negative, the responses to test and prepulses decreased with similar time constants, suggesting a real inactivation process.

Animals↗