76th ENMC International Workshop: pathophysiology and therapy in the mdx mouse 21-23 January 2000, Naarden, The Netherlands.
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Animals↗
Biomedical subjects
R Rüdel
Publications and source records attributed to R Rüdel.
CSF filtration is an effective treatment of Guillain-Barré syndrome: a randomized clinical trial.
OBJECTIVE: To compare CSF filtration (CSFF) and plasma exchange (PE) in the treatment of patients with Guillain-Barré syndrome (GBS). METHODS: In a prospective controlled clinical trial, 37 patients with acute GBS were randomized to receive either CSFF or PE. Inclusion criteria were fulfillment of National Institute of Neurological and Communicative Disorders and Stroke criteria and disability to walk >5 m unassisted. RESULTS: With similar baseline features in both groups (initial disability grades on the six-point grading scale of the GBS Study Group) the primary outcome variable (improvement within 28 days after randomization) was almost identical (test for equivalence p = 0.0014), the mean grade values being 0.82 in the CSFF group and 0.80 in the PE group. After 56 days, 56% (9 of 16 patients) of the CSFF group and 37% (7 of 19 patients) of the PE group had reached grade 2 (i.e., ability of unassisted walking >5 m). After 6 months, the probability to reach grade 2 was about 80% in both groups. In the CSFF group, transient pleocytosis occurred without apparent clinical complications. Clinically relevant complications were higher in the PE-treated group. CONCLUSIONS: Although the number of patients was small, the authors found that the treatment of GBS with CSFF is at least as effective as with PE. CSFF might work by removing from the CSF inflammatory mediators, autoantibodies, or other factors.
Adult↗
Staircase-like potentiation of calcium release in mouse myotubes during repetitive short-term application of threshold caffeine.
The release of Ca2+ in response to caffeine at threshold concentration (5 mM) was studied in mouse skeletal myotubes. Repeated 5-s applications of caffeine, each followed by a 30-s washout, caused Ca2+ releases of consecutively growing amplitude (staircase phenomenon). Each response declined rapidly and had a slow tail. Repeated applications of threshold caffeine lowered the threshold concentration. The interval between threshold applications could be increased to 30 min without loss of potentiation. When threshold caffeine was applied continuously for up to 10 min, the increase in Ca2+ concentration as seen with staircase potentiation did not occur. Depolarization by elevated [K+] or by voltage-clamp steps potentiated caffeine-induced Ca2+ release rapidly as compared to the slow exponential growth of staircase-like potentiation. Gd3+ prevented the depolarization-induced potentiation, but not the staircase phenomenon. Staircase-like potentiation of Ca2+ release was evident even when the voltage sensors were clamped in their resting state; in contrast, potentiated Ca2+ release and its rapid termination apparently require conversion of the voltage sensors to an activated state. Staircase potentiation was blocked when Ca2+ was omitted from the bath, thus pinpointing the source of Ca2+. We suggest that staircase-like potentiation is conditioned by a caffeine-dependent Ca2+ influx across the plasma membrane.
Animals↗
Topology of the human skeletal muscle chloride channel hClC-1 probed with hydrophilic epitope insertion.
To investigate the membrane topology of the skeletal muscle chloride channel ClC-1, we inserted the small antigenic flag (DYKDDDDK) and/or HSV (QPELAPEDPED) epitope tags into nine predicted extra- and intracellular loops along the channel protein. Functional integrity of the modified proteins was tested by measuring the chloride currents conducted by these channels expressed in tsA201 cells. Insertion of the tags into the linkers D1D2, D4D5, D6D7, D8D9 or D11D12 did not alter channel function significantly, whereas insertion into D3D4, D5D6, D9D10 and D10D11 led to loss of function. Intra- or extracellular localisation of the tags was determined by immunofluorescent staining of intact and permeabilised tsA201 cells transiently transfected with the functional epitope-inserted constructs. Intact cells stained for the epitope tags inserted into D1D2, D6D7 and D8D9, indicating that these linkers face the extracellular side of the membrane. No conclusions could be drawn for the location of D4D5 and D11D12. Insertion of the flag epitope at position P260 (linker D4D5), a putative pore-lining region, did not change any of the channel function properties markedly, suggesting that the region surrounding P260 cannot directly line the ion conduction pathway of ClC-1.
Cell Line, Transformed↗
[Wilhelm Erb, founder and first president of the German Society of Neural Medicine].
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Germany↗
Optical detection of mitochondrial NADH content in intact human myotubes.
Time-gated fluorescence spectroscopy in combination with non-radiative energy transfer was used on intact human skeletal myotubes for the determination of the mitochondrial NADH content which is considered to be a sensitive indicator of mitochondrial function. To mimic dysfunction of the mitochondrial energy metabolism, complexes I or III of the respiratory chain were inhibited by drugs. In the absence of the fluorescent mitochondrial marker rhodamine (R123), the NADH autofluorescence (i.e. a signal monitoring cytoplasmic plus mitochondrial NADH) remained unchanged upon inhibition of complex I by rotenone, and was increased by a factor of 2 upon inhibition of complex III by antimycin. In the presence of R123, the autofluorescence of NADH was reduced indicating non-radiative energy transfer from NADH to R123. The ratio of the R123 fluorescence signals obtained with the two excitation wavelengths of 355 nm and 488 nm was taken as a measure of mitochondrial NADH. Relative NADH changes were estimated in the presence of the above-mentioned inhibitors. Upon complex I inhibition, mitochondrial NADH was increased by a factor of 1.5. Upon inhibition of complex III, mitochondrial NADH was increased by a factor of 2. We conclude that time-gated spectroscopy combined with non-radiative energy transfer is an appropriate tool for probing mitochondrial enzyme complex deficiencies.
Antimycin A↗
The small sodium-channel blocking factor in the cerebrospinal fluid of multiple sclerosis patients is probably an oligopeptide.
An endogenous factor that is able to reduce the fast transient sodium current of excitable cells has been reported to exist in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. This was confirmed with nine clinically definite MS patients in the acute relapse. In order to purify and chemically identify the factor, microconcentration and gel filtration high-performance liquid chromatography (HPLC) were applied. After each purification step the activity-containing fraction was determined using a biological assay. With all CSFs the activity was contained in the fraction corresponding to 600-800 Da molecular weight, indicating that the factor is chemically homogeneous. The biological activity of the CSF specimens was not correlated to the laboratory CSF data; however, it was correlated to the area under the 210 nm UV light absorption peak in the corresponding chromatogram, i.e. the 600-800 Da MW fraction. As the factor was degradable by acid hydrolysis and a carboxypeptidase, it is suggested that it might be a small peptide.
Adolescent↗
Anti-GM1 antibodies can block neuronal voltage-gated sodium channels.
Anti-GM1 antibodies, frequently found in the serum of patients with Guillain-Barré syndrome (GBS), have been suggested to interfere with axonal function. We report that IgG anti-GM1 antibodies, raised in rabbits, can reversibly block the voltage-gated Na(+) channels of nerve cells, thus causing a reduction of the excitatory Na(+) current. The block was, however, only substantial when the antibodies were applied together with rabbit complement factors. A solution containing anti-GM1 sera (dilution 1:100) and complement (1:50) reduced the Na(+) current to 0.5 +/- 0.2 times control (mean value +/- SD). Applications of the antibody by itself, complement by itself, or anti-GM2 or anti-GM4 antibodies (1:100) plus complement had little effect. The complexes of anti-GM1 antibodies and complement factors block the ion-conducting pore of the channel directly. In addition, they increase the fraction of channels that are inactivated at the resting potential and alter channel function by changing the membrane surface charge. The described effects may be responsible for conduction slowing and reversible conduction failure in some GBS patients.
Animals↗
Ten years of ENMC--from a patients' initiative to a successful European research institution: the story of the European Neuromuscular Centre.
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Academies and Institutes↗
An endogenous pentapeptide acting as a sodium channel blocker in inflammatory autoimmune disorders of the central nervous system.
Reversible blockade of sodium channels by endogenous substances has been claimed to account for the fast exacerbations and relapses commonly seen in demyelinating autoimmune diseases. Evidence has been provided that in the cerebrospinal fluid of patients with multiple sclerosis or Guillain-Barré syndrome, a sodium-channel-blocking factor exists that has properties of local anesthetic agents. This factor could contribute to the nerve conduction block and paresis seen in these disorders. We describe here a previously unknown endogenous substance in human cerebrospinal fluid with distinct channel-blocking properties even at very low (0.00001 M) concentrations. The pentapeptide with the sequence Gln-Tyr-Asn-Ala-Asp exerted its blocking action by shifting the steady-state inactivation curve of the sodium channels to more-negative potentials, as most local anesthetics do. In the cerebrospinal fluid of healthy individuals, its concentration was about 3 microM, whereas in patients with multiple sclerosis and Guillain-Barré syndrome, it increased 300-1,400%. At these concentrations, the peptide's blocking efficacy was higher than that of 50 microM lidocaine. At a concentration of 10 microM, lidocaine is able to 'unmask' subclinical lesions in multiple sclerosis; thus, the endogenous pentapeptide may well contribute to the fast changes of symptoms. Furthermore, it may become valuable as a marker of disease activity.
Cerebrospinal Fluid↗
Cellular uptake and efficacy of antisense oligonucleotides against RNAs of two Na(+) channel isoforms.
The effects of 15-mer phosphorothioate antisense oligodeoxynucleotides (aODNs) specifically designed against the RNAs of either of two closely related Na(+) channel isoforms, hSkM1 or hH1, were tested in human myotubes. Fluorescence (3'-fluorescein isothiocyanate) labeling showed that mere incubation of cultures with aODNs did not result in aODN uptake, but liposome-mediated transfer was successful and resulted in cytoplasmic and nuclear localization of ODNs. Intracellular fluorescence was stable for at least 3 days. At 5 microM, the hH1-specific aODN was effective in suppressing ion channel function, but the hSkM1-specific aODN was not. Reverse transcription-polymerase chain reaction gave corresponding results on the mRNA level. However, in HEK-293 cells stably expressing hSkM1, the same hSkM1-specific aODN was able to reduce Na(+) currents (2.4 +/- 0.5 nA, n = 11; controls: 6.5 +/- 1.0 nA, n = 14). We conclude that cellular uptake of aODNs and intracellular access to the RNA target are necessary, but not always sufficient conditions for an effective block of mRNA translation in intact cells.
Humans↗
A common mutation (epsilon1267delG) in congenital myasthenic patients of Gypsy ethnic origin.
OBJECTIVE: Mutation analysis of the acetylcholine receptor (AChR) epsilon subunit gene in patients with sporadic or autosomal recessive congenital myasthenic syndromes (CMS). BACKGROUND: The nicotinic AChR of skeletal muscle is a neurotransmitter-gated ion channel that mediates synaptic transmission at the vertebrate neuromuscular junction. Mutations in its gene may cause congenital myasthenic syndromes. A recently described mutation in exon 12 of the AChR epsilon subunit (epsilon1267delG) disrupts the cytoplasmic loop and the fourth transmembrane region (M4) of the AChR epsilon subunit. METHODS: Forty-three CMS patients from 35 nonrelated families were clinically classified as sporadic cases of CMS (group III according to European Neuromuscular Centre consensus) and were analyzed for epsilon1267delG by PCR amplification and sequence analysis. RESULTS: The authors report the complete genomic sequence and organization of the gene coding for the epsilon subunit of the human AChR (accession number AF105999). Homozygous epsilon1267delG was identified in 13 CMS patients from 11 independent families. All epsilon1267delG families were of Gypsy or southeastern European origin. Genotype analysis indicated that they derive from a common ancestor (founder) causing CMS in the southeastern European Gypsy population. Phenotype analysis revealed a uniform pattern of clinical features including bilateral ptosis and mild to moderate fatigable weakness of ocular, facial, bulbar, and limb muscles. CONCLUSIONS: The mutation epsilon1267delG might be frequent in European congenital myasthenic syndrome patients of Gypsy ethnic origin. In general, patients (epsilon1267delG) were characterized by the onset of symptoms in early infancy, the presence of ophthalmoparesis, positive response to anticholinesterase treatment, and the benign natural course of the disease.
Adolescent↗
Identification of three cysteines as targets for the Zn2+ blockade of the human skeletal muscle chloride channel.
Currents through the human skeletal muscle chloride channel hClC-1 can be blocked by external application of 1 mM Zn2+ or the histidine-reactive compound diethyl pyrocarbonate (DEPC). The current block by Zn2+ strongly depends on the external pH (pKa near 6.9), whereas the block by DEPC is rather independent of the pH in the range of 5.5 to 8.5. To identify the target sites of these reagents, we constructed a total of twelve cysteine- and/or histidine-replacement mutants, transfected tsA201 cells with them, and investigated the resulting whole-cell chloride currents. The majority of the mutants exhibited a similar sensitivity toward Zn2+ or DEPC as wild type (WT) channels. Block by 1 mM Zn2+ was nearly absent only with the mutant C546A. Four mutants (C242A, C254A, H180A, and H451A) were slightly less sensitive to Zn2+ than WT. Tests with double, triple, and quadruple mutants yielded that, in addition to C546, C242 and C254 are also most likely participating in Zn2+-binding.
Cell Line↗
Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers.
1. Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic-dystrophic double mutant ADR-MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2. Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura-2, were slightly higher in MDX (63 +/- 20 nM; means +/- s.d.; n = 454 analysed fibres) and ADR-MDX (65 +/- 12 nM; n = 87) fibres than in controls (51 +/- 20 nM; n = 265). 3. The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50 % of MDX fibres (n = 68), but in only 20 % of controls (n = 54), the decay time constants were > 35 ms. 4. Bath application of Mn2+ resulted in a progressive quench of fura-2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 +/- 1.9 % min-1; n = 275) than in controls (2.03 +/- 1.4 % min-1; n = 204). The quench rate in ADR-MDX fibres (2.49 +/- 1.4 % min-1; n = 87) was closer to that of controls. 5. The Mn2+ influx into MDX fibres was reduced to 10 % by Gd3+, to 19 % by La3+ and to 47 % by Ni2+ (all at 50 microM). Bath application of 50 microM amiloride inhibited the Mn2+ influx to 37 %. 6. We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR-MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability.
Amiloride↗
Regulation of the human skeletal muscle chloride channel hClC-1 by protein kinase C.
1. The regulation of a recombinant human muscle chloride channel, hClC-1, by protein kinase C (PKC) was investigated in human embryonic kidney (HEK 293) cells. 2. External application of 4beta-phorbol esters (4beta-PMA) reduced the instantaneous whole-cell current amplitude over the entire voltage range tested. This effect was abolished when the cells were intracellularly perfused with a specific protein kinase C inhibitor, chelerythine. Inactive 4alpha-phorbolesters did not affect the chloride currents. We conclude that the effect of 4beta-phorbol esters is mediated by protein kinase C (PKC). 3. Activation of PKC resulted in changes in macroscopic current kinetics. The time course of current deactivation determined in the presence and absence of 4beta-phorbol esters could be fitted with the sum of two exponentials and a constant value. In the presence of phorbol esters, the fast time constants and the minimum value of the fraction of non-deactivating current were increased, whereas the voltage dependence of all fractional current amplitudes remained unchanged. PKC-induced phosphorylation had only small effects on the voltage dependence of the relative open probability and the maximum absolute open probability was unaffected by treatment with 4beta-PMA, as shown by non-stationary noise analysis. 4. The kinetic changes indicate that phosphorylation alters functional properties of active channels. Since the absolute open probability is not reduced, the observed macroscopic current reduction implies alterations of the ion permeation process. 5. Phosphorylation by PKC appears to affect ion transfer and gating processes. It is postulated that the phosphorylation site may be located at the cytoplasmic vestibule face of the pore.
Alkaloids↗
Electrical myotonia in heterozygous carriers of recessive myotonia congenita.
We investigated electrophysiologically the unaffected parents of patients with recessive myotonia congenita. We studied 18 families, in nine of which the diagnosis was confirmed by molecular genetics. Brief myotonic discharges were present in at least one parent in 67% of the families. Fathers were more likely than mothers to show these discharges. The difficulty in distinguishing very mildly affected parents with dominant myotonia congenita from the heterozygous carriers of recessive myotonia congenita is stressed.
Action Potentials↗
A small sodium channel blocking factor in the cerebrospinal fluid is preferentially found in Guillain-Barré syndrome: a combined cell physiological and HPLC study.
The cerebrospinal fluid (CSF) of patients with Guillain-Barré syndrome (GBS) contains a low molecular weight factor with sodium channel blocking activity. This study investigated whether such activity also exists in the CSF of patients with other neurological diseases. Further, using high-performance liquid chromatography (HPLC) we tested whether the electrophysiological effect of the CSF is correlated with the size of the corresponding peak in the chromatograms. The existence of sodium channel blocking activity was tested in 27 native CSF samples of three groups of patients (group 1: GBS, n = 13; group 2: other inflammatory diseases, n = 8; group 3: controls, n = 6). NH15-CA2 neuroblastoma x glioma cells in the whole-cell recording configuration was used as a system for assaying the sodium channel blocking activity of CSF specimens. CSF shifted the steady-state inactivation curve of the sodium channels reversibly by -10.2 +/- 4.4 mV in group 1, -6.7 +/- 3.9 mV in group 2, and - 3.5 +/- 2.8 mV in group 3 (P < 0.01). The shift was greater in demyelinating (9.3 +/- 4.7 mV) than in nondemyelinating (5.6 +/- 3.9 mV) diseases (P < 0.04). HPLC analysis of CSFs showed a well separated peak containing the substance responsible for the electrophysiological effect at about 41 min elution time. The peak covered the molecular weight range of 600-800 Da. Sodium channel blocking activity of CSFs and areas of the corresponding peak in the chromatograms were well correlated. We conclude that sodium current inhibition by a low molecular weight factor is generally present but increased in GBS.
Cerebrospinal Fluid↗
A transient and a persistent calcium release are induced by chlorocresol in cultivated mouse myotubes.
The effect of 4-chloro-m-cresol (4-CmC), a stabilizing agent used in commercial preparations of the muscle relaxant succinylcholine, on intracellular free calcium levels in cultivated mouse myotubes was studied. Calcium signals were monitored with an inverted microscope equipped for fluorescence photometry using fura-2 as the calcium indicator. Upon bath application of 500 microM 4-CmC for 90 s, two separate calcium signals, a transient and a sustained one, could be regularly discriminated. First, with a delay of 2 s, the intracellular calcium concentration increased from 41+/-13 to 541+/-319 nM, peaked after 2-5 s and declined within 10 s to nearly resting values (n=36). Then, after a delay of up to 20 s, intracellular calcium rose quickly again to almost the same value and stayed elevated as long as the drug was applied. Upon drug removal, intracellular calcium rapidly decreased to a new level that was always slightly higher than the original base line. At 250 microM 4-CmC, the response was small, whereas at 500 microM it was at its maximum. Thus, the concentration-response curve was very steep. Replacement of extracellular calcium by EGTA and application of calcium channel blockers revealed that, for both the transient and the sustained response, calcium was released from intracellular stores. Pre-treatment with thapsigargin (0.1 microM) or ryanodine (10 microM) abolished both signal components. Repeated short-term applications of 4-CmC suggest that the two components may arise from different systems.
Animals↗