Heinrich Brinkmeier

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 μm). Bath application of 50 μm 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.

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.

Intracellular Ca2+ concentrations are not elevated in resting cultured muscle from Duchenne (DMD) patients and in MDX mouse muscle fibres

The free intracellular calcium concentration, [Ca2+]i, was studied in single myotubes using the fluorescent Ca2+ indicator fura-2. Myotubes cultured from satellite cells of small muscle specimens from Duchenne muscular dystrophy (DMD) patients were compared with human control myotubes and with myotubes cultured from MDX and control mouse muscle satellite cells. The resting [Ca2+]i levels in DMD and control myotubes were not significantly different, i. e. 104 ±26 nM (mean ± SD, n=190 cells from eight DMD patients) compared with 97±25 nM (175/seven controls) and were not significantly lower than the corresponding murine values (154±33 nM, n=135 MDX myotubes; 159±34 nM, n=135 controls). All myotubes reacted to 10 μM acetylcholine or 40 mM KCl with fast transient increases of [Ca2+]i. After application of a hyposmotic (130 mOsm) solution, [Ca2+]i was increased 1.5- to 3-fold within 2–3 min, the DMD myotubes tending to stronger reactions (significantly higher [Ca2+]i in 2 out of 6 cases). The response was usually transient, [Ca2+]i decreasing to the initial level within 10 min. Gadolinium (50 μM) reduced the response by 50%–70%, indicating that the osmotic shock increased Ca2+ influx. During exposure to high (15 mM) [Ca2+]e, [Ca2+]i of DMD and control cells was 1.5- to 2-fold higher. Adult muscle fibres from MDX mice and controls showed identical Ca2+ resting levels (n=45 fibres from three mice in each case), but did not respond to decreased external osmolarity with a change in [Ca2+]i. The results indicate that lack of dystrophin in muscle fibres does not necessarily lead to increased [Ca2+]i. It is suggested that increased [Ca2+]i is probably a secondary consequence of fibre damage.

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 μM, whereas in patients with multiple sclerosis and Guillain-Barré syndrome, it increased 300–1,400%. At these concentrations, the peptides blocking efficacy was higher than that of 50 μM lidocaine. At a concentration of 10 μM, 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.

The acute paralysis in Guillain-Barré syndrome is related to a Na+ channel blocking factor in the cerebrospinal fluid

The effect of cerebrospinal fluid (CSF) from patients with severe polyradiculoneuritis (Guillain-Barré syndrome, GBS) on voltage-dependent Na+ channels of myoballs was studied. The transient Na+ currents, elicited by repetitive stimulation at 1 Hz, were inhibited by the CSF from most of the GBS patients to 10%–40% the control value. The inhibition was complete in about 5 s and was fully reversible. Such inhibition was never seen with control CSF. The blocking property of the CSF from GBS patients was lost after the number of cells and the protein content had been lowered by means of a clinical filtration technique for cerebrospinal fluid. The results demonstrate that in Guillain-Barré syndrome blocking factors of Na+ channels are present in the CSF, impairing neuron impulse conduction, and thereby causing muscular weakness and sensory disturbances in the affected patient.

ACTIVATORS OF PROTEIN-KINASE-C INDUCE MYOTONIA BY LOWERING CHLORIDE CONDUCTANCE IN MUSCLE

Certain phorbol esters and bryostatin 1, known activators of protein kinase C, were found to induce, in genetically normal muscle, an electrical membrane instability leading to unscheduled series of action potentials. Like in hereditary myotonias of man, goat and mouse, these symptoms were caused by a drastically lowered sarcolemmal chloride conductance. Our results indicate that the chloride channels of mammalian muscle may be subject to modulation by the protein kinase C-diacylglycerol system.

Interleukin-2 inhibits sodium currents in human muscle cells

The effect of the T cell growth factor interleukin-2 (IL-2) on muscular Na+ channels was studied in myoballs produced from primary human muscle cultures. The transient Na+ inward currents of the myoballs, elicited by repetitive stimulation at 1 Hz and recorded in the whole-cell mode, were inhibited by IL-2 applied to the external solution, the half maximum effect occurring at 300 U/ml. The effect was complete within 5 s and was totally reversible, the on and off effects having identical time courses. The h∞ curve was shifted in negative direction indicating that the mechanism of IL-2 action is a conversion of the Na+ channels into a state of inactivation. The reaction of the IL-2 solution with an anti IL-2 antibody neutralized the inhibitory effect on the Na2+ currents, indicating a specific effect of the peptide growth factor interleukin-2 on muscular Na+ channels. The connection of IL-2 and Na+ channels may be important in inflammatory processes of muscle and nerve.

Factors in the cerebrospinal fluid of multiple sclerosis patients interfering with voltage-dependent sodium channels

The effect of cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) on voltage-dependent Na+ channels in human myoballs was studied. The transient Na+ currents, elicited by whole-cell depolarization from -85 to -20 mV, were decreased to 75-25% the control value in the presence of CSF from all 7 MS patients investigated. The effect was complete in about 5 s and was fully reversible on admission of standard external fluid. Such decrease was not or only to a minor extent observed with 10 out of 11 control CSFs from patients without inflammatory neurological disease. The origin of the factors interfering with the Na+ channels is unknown. It is suggested that, in addition to demyelination, impaired Na+ channel function might cause the symptoms in MS.

Specific modifications of the membrane fatty acid composition of human myotubes and their effects on the muscular sodium channels

The fatty acid (FA) composition of human myotube primary cultures was varied by modifications of the contents of FA in the culture medium. An incubation time of 18 h with a defined FA mixture resulted in the most effective alteration of the original FA pattern of the cells. The increases reached for the relative amounts of palmitic acid (16:0), linoleic acid (18:2) or arachidonic acid (20:4) were 3-5-fold. More than 50% of the extra FA were incorporated in the phospholipid fraction, the remaining share in the triglyceride fraction. Shorter incubation times resulted in less FA incorporation, longer incubation times raised the uptake of FA into the triacylglycerol fraction. For a study of the influence of the membrane modification on the function of the sodium channels, the myotubes were converted into myoballs. The sodium channel properties were then determined using the whole-cell clamp technique. The modified cultures showed no significant alterations in the time constants of activation and inactivation, in the voltage dependence of inactivation (h infinity curves) or in the average amplitudes of the sodium currents.

Local anaesthetic-like effect of interleukin-2 on muscular Na+ channels: no evidence for involvement of the IL-2 receptor

The effects of interleukin-2 (IL-2) on muscular Na+ currents were studied in human myoballs. The transient Na+ inward currents, elicited by repetitive depolarizations at 1 Hz and recorded in the whole-cell mode, were inhibited by the cytokine, the half-maximum effect occurring at about 500 U/ml. The effects resembled those of local anaesthetics without use dependence, as the inactivation (h∞) curve was shifted in a negative direction while the current/voltage curve was not affected. As with these local anaesthetics, depolarization at 1, 4 and 8 Hz in the presence of IL-2 did not produce any cumulative block. The interaction of IL-2 with the Na+ channels is very fast (within ms) and it is suggested that it occurs when the Na+ channels are in the state of fast inactivation. The recovery from inactivation was only slightly slowed by IL-2, in agreement with the absence of any use dependence. All effects were readily reversible on washout of the cytokine. The effects were seen both in tetrodotoxin-(TTX)-sensitive adult Na+ channels and in TTX-insensitive juvenile channels. In contrast to the whole-cell configuration, no inhibition was visible in the attached-patch configuration. Further, the preincubation with an anti-IL-2-receptor antibody did not prevent the inhibitory effect of IL-2 on the Na+ currents. It is concluded that the cytokine blocks the voltage-dependent muscular Na+ channels by keeping the channels in the state of fast inactivation. An IL-2 receptor and a second messenger system are not likely to be involved in this reaction.