Adriana S. Losavio

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.

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.

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved.

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.

The effect of aminophylline on the contraction threshold of rat diaphragm fibers and its modification by 9-aminoacridine

We studied the effect of aminophylline (1mM) and 9-aminoacridine (100 microM) on the contraction threshold (CT) of rat diaphragm fibers (25 degrees C). The CT was measured by direct visualization (200 X) of the fiber under current-clamp conditions. The main findings are the following: 1) Aminophylline lowers the CT toward more negative values of the resting membrane potential (Vm). 2) 9-aminoacridine, a drug that diminishes Ca2+ release from the sarcoplasmic reticulum (SR), shifts the CT toward more positive values: 3) this effect is overcome by aminophylline. We suggest that the displacement in the CT to more negative Vm plays an important role in the potentiating effect of aminophylline. This could be the result of an enhancement of Ca2+ release from the SR.

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.

Blockers of potassium current and resting membrane potential in rat muscle fibers

Rat diaphragm fibers were equilibrated for several hours 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 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.

Endogenous purines modulate K+-evoked ACh secretion at the mouse neuromuscular junction

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co‐released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y13 and A1, A2A, and A3 receptors, respectively. To elucidate the action of endogenous purines on K+‐dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end‐plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K+, the P2Y13 antagonist N‐[2‐(methylthio)ethyl]‐2‐[3,3,3‐trifluoropropyl]thio‐5′‐adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR‐C69931MX) increased asynchronous ACh secretion while the A1, A3, and A2A antagonists 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), (3‐Ethyl‐5‐benzyl‐2‐methyl‐4‐phenylethynyl‐6‐phenyl‐1, 4‐(±)‐dihydropyridine‐3,5‐, dicarboxylate (MRS‐1191), and 2‐(2‐Furanyl)‐7‐(2‐phenylethyl)‐7H‐pyrazolo[4,3‐e][1,2,4]triazolo[1,5‐c]pyrimidin‐5‐amine (SCH‐58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S‐(p‐nitrobenzyl)‐6‐thioinosine provoked a reduction of 10 mM K+‐evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K+, endogenous ATP/ADP and adenosine bind to inhibitory P2Y13 and A1 and A3 receptors since AR‐C69931MX, DPCPX, and MRS‐1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto‐5′‐nucleotidase inhibitor α,β‐methyleneadenosine 5′‐diphosphate sodium salt. SCH‐58261 only reduced neurosecretion at 20 mM K+, suggesting that more adenosine is needed to activate excitatory A2A receptors. At high K+ concentration, the equilibrative transporters appear to be saturated allowing the accumulation of adenosine in the synaptic cleft. In conclusion, when motor nerve terminals are depolarized by increasing K+ concentrations, the ATP/ADP and adenosine endogenously generated are able to modulate ACh secretion by sequential activation of different purinergic receptors.

Differential effect of serum from bipolar versus schizophrenic patients on spontaneous acetylcholine release at mammalian neuromuscular junction

OBJECTIVE The diagnosis of bipolar disease frequently requires a long time since the age of onset, especially because the disease is misdiagnosed with schizophrenia. The aim of the present work was to investigate whether sera from bipolar patients have an active substance that allows making a fast identification of the disease. METHODS Sera from healthy volunteers, euthymic and non-stabilized bipolar patients, and schizophrenic patients were passively transferred into CF1 mice and after 2 day injections, MEPP frequency from diaphragm muscles was recorded. The same procedure was performed with sera fraction of high and low MW (cut-off 3000). RESULTS Sera from non-stabilized bipolar patients induced a decreased MEPP frequency and occluded the presynaptic inhibitory effect of the specific adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) in the recipient mice, while in the euthymic bipolar group spontaneous secretion reached control values although the action of CCPA was still prevented. Similar results were obtained with low MW sera fraction from euthymic and non-stabilized bipolar patients. The addition of adenosine deaminase to the sera fraction prevented the modification of spontaneous ACh release. In mice injected with sera from schizophrenic patients, MEPP frequency was within control values and CCPA induced its typical inhibitory action. CONCLUSIONS These results indicate that bipolar patients contain in their blood an active substance compatible with adenosine, which was able to modify spontaneous ACh release in the recipient mice. This effect was not observed with sera from healthy volunteers and schizophrenic patients. The increase of adenosine concentration may result from synaptic hyperactivity that presumably plays a role in the symptoms of bipolar disorder and/or may derive from peripheral cells through a more general mechanism. SIGNIFICANCE The different results obtained with bipolar and schizophrenic sera raise the possibility that the passive transfer model could be used as a diagnostic test in the future.