S. Muchnik

Long-term follow-up of Lambert-Eaton syndrome treated with intravenous immunoglobulin

Recent reports have shown that patients with Lambert‐Eaton myasthenic syndrome (LEMS) improve transiently after high‐dose intravenous immunoglobulin (IVIG) administration. Information about the usefulness of IVIG for long‐term treatment is rather scanty. Our findings demonstrate the efficacy of monthly IVIG courses at a dose of 0.4 g/kg/day for 5 days, in a 41‐year‐old patient with LEMS without detectable malignancy. Improvement in limb strength, peak expiratory flow rate, and electrophysiological parameters, as well as clinical signs following IVIG, was evident as early as 7 days after the first course and is still maintained at 24‐months follow‐up.

Presynaptic inhibition of spontaneous acetylcholine release induced by adenosine at the mouse neuromuscular junction

At the mouse neuromuscular junction, adenosine (AD) and the A1 agonist 2‐chloro‐N6‐cyclopentyl‐adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A1 AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage‐dependent calcium channels (VDCCs) associated with tonic secretion (L‐ and N‐type VDCCs), we measured the miniature end‐plate potential (mepp) frequency in mouse diaphragm muscles. Blockade of VDCCs by Cd2+ prevented the effect of the CCPA. Nitrendipine (an L‐type VDCC antagonist) but not ω‐conotoxin GVIA (an N‐type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L‐type VDCCs only. As A1 receptors are coupled to a Gi/o protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N‐(2[p‐bromocinnamylamino]‐ethyl)‐5‐isoquinolinesulfonamide (H‐89, a PKA inhibitor), nor 1‐(5‐isoquinolinesulfonyl)‐2‐methyl‐piperazine (H‐7, a PKC antagonist), nor phorbol 12‐myristate 13‐acetate (PHA, a PKC activator) modified CCPA‐induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. The effect of CCPA was eliminated by the calmodulin antagonist N‐(6‐aminohexil)‐5‐chloro‐1‐naphthalenesulfonamide hydrochloride (W‐7) and by ethylene glycol‐bis(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester ɛ6TΔ‐BM, which suggests that the action of CCPA to modulate L‐type VDCCs may involve Ca2+‐calmodulin. To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K+ concentrations. The effect of CCPA on ACh secretion evoked by 10 mM K+ was prevented by the P/Q‐type VDCC antagonist ω‐agatoxin IVA. CCPA failed to inhibit the increases in mepp frequency evoked by 15 and 20 mM K+. We demonstrated that, at high K+ concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX, an A1 receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20 mM K+ in the absence of the drugs. Moreover, CCPA was able to induce presynaptic inhibition in the presence of ADA. It is concluded that, at high K+ concentrations, the activation of A1 receptors by endogenous AD prevents excessive neurotransmitter release.

Mechanical and electrical properties of denervated rat skeletal muscles

Mechanical activity (twitch and tetanus) and electrical activity (single and repetitive action potentials) were recorded in vitro (34 degrees C) in control and denervated (3 to 14 days) soleus and extensor digitorum longus muscles of the rat. After denervation tetanic tension (100 to 200 Hz, 500 ms duration) was decreased in both types of muscles. Denervation reduced significantly the rates of rise and fall and the amplitude of the action potential in both types of muscle fibers. In denervated fibers with very low resting membrane potential no action potentials could be recorded: in these fibers only a slow response without overshoot was detected. Hyperpolarization of denervated fibers to -90 mV prior to application of the depolarizing pulse increased their excitability. Action potential amplitudes were well maintained during tetanic stimulation (200 Hz, 40 to 90 ms) in innervated fibers. Depolarization of the innervated fibers with cathodic current before the tetanic pulse hindered the generation of repetitive action potentials at 200 Hz. A proportion of denervated fibers stimulated at 100 to 200 Hz generated only one action potential or gave rise to an incomplete train. Hyperpolarization of the denervated fibers resulted in an improvement in the ability to generate a train of action potentials at 100 to 200 Hz. A group of denervated fibers exhibited well maintained action potentials during tetanus. We suggest that failure in the repetitive electrical activity of denervated fibers could be the reason for the reduced tension of tetanus. Depolarization of the fibers and/or the increment in the electrical time constant of the sarcolemma are suggested for the decrease in the electrical excitability of denervated fibers.

Facilitation of spontaneous acetylcholine release induced by activation of cAMP in rat neuromuscular junctions.

Regulation of neurotransmitter release is thought to involve modulation of the release probability by protein phosphorylation. Activation of the cAMP-protein kinase A (PKA) pathway has been shown to facilitate synaptic transmission in mammalian neuromuscular synapses, although the relevant phosphorylation targets are mostly unknown. We found that the inhibitor of the phosphodiesterase aminophylline (1 mM AMIN), the membrane-permeable analog of cAMP, 8-Br-cAMP (5 mM) and, the direct adenylate cyclase activator, forskolin (20 microM), induced an increase of miniature end-plate potentials (MEPPs) frequency in rat neuromuscular junctions. We investigated the possible involvement of the voltage-dependent calcium channels (VDCC), since these proteins are known to be phosphorylated by PKA. But this possibility was ruled out, since the increase in MEPPs frequency was not attenuated by the VDCC blocker Cd2+ (100 microM) and it was observed when AMIN was studied on hyperosmotic response, which is independent of [Ca2+]o and of Ca2+ influx through the VDCC. The lack of action of AMIN on MEPPs frequency when [Ca2+]i was diminished by exposing the preparations to zero Ca2+-EGTA solution (isotonic condition) or when nerve terminals were loaded with a permeant Ca2+ chelator (BAPTA-AM) (hypertonic condition), indicate that cAMP-mediated presynaptic facilitation is a function of nerve terminal Ca2+ concentration. We also found that AMIN exerted a comparable increase in MEPPs frequency in control and high K+ (10 and 15 mM), suggesting a single mechanism of action for spontaneous and K+-induced secretion.

Effect of amyotrophic lateral sclerosis serum on calcium channels related to spontaneous acetylcholine release

OBJECTIVES The aim of this work was to further investigate the effect of sera from sporadic amyotrophic lateral sclerosis (ALS) patients on miniature end-plate potentials (MEPP) frequency, by the mouse passive transfer model, and to study whether the transferred serum induces any change in the sensitivity of the L-type voltage-dependent calcium channels (VDCC) to its specific blocker Nitrendipine. METHODS A total of 35 CF1 mice were divided into 3 groups: (a) ALS group receiving sera from 15 patients that had been clinically and electromyographycally diagnosed as having sporadic ALS; (b) normal group receiving sera from 13 healthy volunteers and from 3 disease control patients, and (c) control group, which was kept untreated. Animals in groups (a) and (b) received daily intraperitoneal injection of 0.5-1ml of serum for 3 days, and 24h later the left hemidiaphragm was excised for electrophysiological recordings. RESULTS Analysis of MEPPs frequency recorded from ALS group showed that 3 of them induced an increase in spontaneous neurotransmitter release while in 4 a decrease was observed, suggesting that sera alter spontaneous secretion as result of an increased or decreased Ca(2+) influx through the normally involved N-type or L-type VDCC, respectively. When the effect of Nitrendipine, an L-type VDCC blocker, was studied on ALS sera-injected mice, we found variable responses to the drug: only two mice showed control sensitivity to Nitrendipine, while in 7 its action was lower and surprisingly in 4 was greater than that without the drug. CONCLUSIONS These results suggest that ALS sera contain factor(s) that are able to modify spontaneous neurotransmitter release by altering calcium current through L-type and N-type VDCC, and even inducing changes in the sensitivity to the L-type VDCC blocker.

Familial congenital facial diplegia: Electrophysiologic and genetic studies

A 13-year-old boy with autosomal-dominant congenital facial diplegia was evaluated by electrophysiologic and genetic investigations. Thirteen members of his family were affected over 4 generations. The electrophysiologic studies revealed blink reflex abnormalities. Both R1 and R2 responses were prolonged on the left side after ipsilateral stimulation, while R2 was also delayed by contralateral stimulation. Ipsilateral R1 and R2 were of normal latencies when the right side was stimulated. A third ipsilateral response at 63 msec of latency could be obtained when stimulating the left side. These findings suggest functional damage to the brainstem. Further support for this interpretation was provided by the prolonged time between waves I and V, bilaterally, documented by study of brainstem auditory evoked potentials.

Effects of antinomycin D on fibrillation activity in denervated skeletal muscles of the rat

Abstract The effects of actinomycin D on fibrillation activity, acetylcholine sensitivity and resting membrane potential of denervated muscles of the rat was studied. Actinomycin D (0.7 mg/kg I.V.) administered 1 day after denervation delays the appearance of fibrillation for approximately 3 days. If this drug is given 5–7 days after denervation, it is also capable of blocking the already established fibrillation but fails to suppress extrajunctional cholinergic receptors and to reverse the fall in resting potential. The mechanical responses of denervated muscles are unaffected by actinomycin D. These results suggest that in fibrillation a genetic induction of newly formed RNA and protein is involved. It is also suggested that these molecules probably have a more rapid turnover than those required for the formation of extrasynaptic receptors in denervated muscle.

Caffeine contractures in denervated slow-twitch and fast-twitch muscles of the rat

Caffeine contractures (25 mM) and twitches were registered in vitro (34 degrees C) in normal and denervated soleus and extensor digitorum longus of the rat. Both muscles lost weight progressively after denervation although the loss of weight in soleus muscles was more manifest. Denervated extensor digitorum longus muscles showed an increase in the caffeine contracture tension (expressed in g/g muscle) whereas the response generated by denervated soleus muscles was smaller than the control values. The time to peak of the caffeine responses was shortened in both types of muscles after denervation. Twitches were larger and had a slower time course than in normal muscles, especially in the extensor digitorum longus muscles. These findings suggest that some changes in the excitation-contraction coupling could be responsible for the potentiation of the twitches in denervated muscles.

Muscle mechanical changes following denervation and their modification by the use of an inhibitor of RNA synthesis

Abstract Mechanical properties of soleus muscles from adult Wistar rats were studied “in vitro”. Contraction time, time for half relaxation, duration of the active state, fusion frequency and tetanus-twitch ratio were measured. In a first group of experiments normal innervated muscles were compared with muscles denervated for ten days. Significative differences in their contractile properties were found. In a second group of rats Actinomycin D was injected intravenously eight days after denervation and soleus contractile parameters compared 48 hours later with those obtained in the same muscle of denervated untreated rats. Treated muscles did not show significative differences with the untreated ones. However, when Actinomycin D was injected at the time of denervation differences in the duration of the active state were detected. It is suggested in the present paper that mechanical changes following denervation may be considered an induced phenomenon, as was demonstrated in other denervatory changes.

Barium resistant potassium current in mammalian skeletal muscle following denervation

Inward rectifier potassium channels are thought to be related to resting membrane potential and in innervated skeletal muscle they are specially sensitive to the blocking action of Ba2+ ions. After denervation other channels are known to become resistant to their blockers. We study the effect of Ba2+ upon the inward rectifier potassium channels after denervation. Rat extensor digitorum longus fibers were equilibrated for 150 minutes in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 35 minutes. This repolarization was blocked by 5 mM BaCl2 in innervated muscles and in muscles denervated for 7 days, but failed to do so after 14 days of denervation. Voltage-clamp experiments performed in lumbricalis denervated muscle showed a lack of effect of Ba2+ upon potassium current after 18 days of denervation. This results suggest that the inward rectifier potassium channels become resistant to Ba2+ ions after denervation, indicating a neural influence.