Jean-Pierre Pennec

Mechanisms of the sensory effects of tacrolimus on the skin

Background  Tacrolimus is an immunosuppressant drug currently used for the treatment of atopic dermatitis and pruritus. This topical therapy is effective and safe, but transient burning, stinging and itch are frequently reported.

Effects of chronic sepsis on the voltage-gated sodium channel in isolated rat muscle fibers.

Objective:Physiopathology of critical illness polyneuromyopathy was investigated in several animal-based models. Electrophysiologic approach was achieved in denervated and corticosteroid-induced myopathy; other models based on sepsis or inflammatory factors (zymosan, cytokines) were also used but did not consider voltage-gated sodium channel implication in neuromuscular weakness. We have studied electrophysiologic effects of chronic sepsis on an intact neuromuscular rat model with special consideration to the subtypes of sodium channels involved. Design:Experimental animal study. Setting:University laboratory. Subjects:Wistar rats. Interventions:Chronic sepsis was achieved by a technique of cecal ligature and needle perforation. Ten days after surgery, the rats were killed. Fast-twitch flexor digitorum brevis was excised and dissociated in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid–buffered saline supplemented with 3.0 mg/mL collagenase. Fast sodium currents were recorded by a macropatch clamp technique at room temperature (22 ± 2°C) in a cell-attached configuration. Measurements and Main Results:A decrease in maximal sodium current and in conductance was evidenced without modification of the sodium Nernst potential. A shift of the voltage inactivation curve toward more negative potentials could explain the observed decrease in excitability. In parallel, we observed an up-regulation of NaV 1.5–type sodium channels. Conclusions:Chronic inflammation and sepsis induced modifications of sodium channel properties that could contribute to muscular inexcitability. This inexcitability can be elicited by a modification of properties or type of voltage-gated sodium channels. Our results lead us to explain this inexcitability by an up-regulation of NaV 1.5 sodium channel.

Effects of chronic sepsis on contractile properties of fast twitch muscle in an experimental model of critical illness neuromyopathy in the rat.

Objective:Critical illness polyneuromyopathy has been extensively studied in various animal models regarding electrophysiological aspects or molecular mechanisms involved in its physiopathology; however, little data are available on its main clinical feature, that is, muscular weakness. We have studied the effects of chronic sepsis in rats with special consideration to contractile and neuromuscular blockade properties in relation with the level of messenger RNA (mRNA) coding for ryanodine and acetylcholine receptors. Design:This was an experimental animal study. Setting:This study was conducted at a university laboratory. Subjects:Subjects consisted of Wistar rats. Interventions:Chronic sepsis was achieved by cecal ligation and needle perforation. Ten days after surgery, fast twitch extensor digitorum longus was excised for extraction and assays of mRNA coding for ryanodine and acetylcholine receptor subunits and contralateral muscle was tested in vivo on a mechanical bench. A fatigability index was measured and neuromuscular blockade properties using atracurium were evaluated. Measurements and Main Results:A decrease in active force developed by extensor digitorum longus associated with an increase in passive force is induced by chronic sepsis. Maximal force at optimal length during twitch contraction was significantly reduced (0.25 ± 0.09 N vs. 0.17 ± 0.06 N); contraction and relaxation speeds were higher as shown by the decrease of respective time constants (3.75 ± 0.01 msec vs. 2.70 ± 0.0 msec, 10.76 ± 0.03 msec vs. 7.62 ± 0.03 msec) in the control group compared with the septic group. Fatigability index was significantly lower (23 ± 0.11% vs. 59 ± 0.19%) in septic rats. These rats also showed quicker blockade and shorter recovery after atracurium administration. Sepsis induced a significant increase of the expression of ryanodine receptor (RyR) RyR1 along with a steady expression of RyR3 mRNA, leading to a 5.6-fold increase of RyR1/RyR3 ratio with a steadiness of mRNA corresponding to acetylcholine-receptors. Conclusions:Chronic inflammation and sepsis induced a decrease in contractile performances of extensor digitorum longus along with accelerated kinetics of atracurium possibly induced by modified expression of RyR1 receptors and not acetylcholine-receptors.

Rat Merkel cells are mechanoreceptors and osmoreceptors.

Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca2+ channels and hypotonic-induced membrane deformation is known to lead to Ca2+ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification. Their properties were investigated through electrophysiological studies. Voltage-gated K+, Ca2+ and Ca2+-activated K+ (KCa) channels were identified, as previously described. Here, we also report the activation of Ca2+ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca2+ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.

Rapid protein kinase C‐dependent reduction of rat skeletal muscle voltage‐gated sodium channels by ciliary neurotrophic factor

The ciliary neurotrophic factor (CNTF), known to exert long‐term myotrophic effects, has not yet been shown to induce a rapid biological response in skeletal muscles. The present in vitro study gives rise to the possibility that CNTF could affect the sodium channel activity implied in the triggering of muscle fibre contraction. Therefore, we investigated the effects of an external CNTF application on macroscopic sodium current (INa) in rat native fast‐twitch skeletal muscle (flexor digitorum brevis, FDB) by using a cell‐attached patch‐clamp technique. The INa peak amplitude measured at a depolarizing pulse from −100 to −10 mV is rapidly reduced in a time‐ and dose‐dependent manner by CNTF (0.01–20 ng ml−1). The maximal decrease is 25% after 10 min incubation in 2 ng ml−1 CNTF. There was no alteration in activation or inactivation kinetics, or in activation curves constructed from current–voltage relationships in the presence of CNTF. In contrast, the relative INa inhibition induced by CNTF is accompanied by a hyperpolarizing shift in the midpoint of the inactivation curves: −6 and −10 mV for the steady‐state fast and slow inactivation, respectively. Furthermore, CNTF induces a 5 mV hyperpolarization of the resting membrane potential of the fibres. The effects of CNTF are similar to those of 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), a protein kinase C (PKC) activator, when no effect is observed in the presence of chelerythrine, a PKC inhibitor. These results suggest that, in skeletal muscle, CNTF can rapidly decrease sodium currents by altering inactivation gating, probably through an intracellular PKC‐dependent mechanism that could lead to decreased membrane excitability. The present study contributes to a better understanding of the physiological role of endogenous CNTF.

Tumor necrosis factor-α downregulates sodium current in skeletal muscle by protein kinase C activation: involvement in critical illness polyneuromyopathy

Sepsis is involved in the decrease of membrane excitability of skeletal muscle, leading to polyneuromyopathy. This effect is mediated by alterations of the properties of voltage-gated sodium channels (Na(V)), but the exact mechanism is still unknown. The aim of the present study was to check whether tumor necrosis factor (TNF-α), a cytokine released during sepsis, exerts a rapid effect on Na(V). Sodium current (I(Na)) was recorded by macropatch clamp in skeletal muscle fibers isolated from rat peroneus longus muscle, in control conditions and after TNF-α addition. Analyses of dose-effect and time-effect relationships were carried out. Effect of chelerythrine, a PKC inhibitor, was also studied to determine the way of action of TNF-α. TNF-α induced a reversible dose- and time-dependent inhibition of I(Na). A maximum inhibition of 75% of the control current was observed. A shift toward more negative potentials of activation and inactivation curves of I(Na) was also noticed. These effects were prevented by chelerythrine pretreatment. TNF-α is a cytokine released in the early stages of sepsis. Besides a possible transcriptional role, i.e., modification of the channel type and/or number, we demonstrated the existence of a rapid, posttranscriptional inhibition of Na(V) by TNF-α. The downregulation of the sodium current could be mediated by a PKC-induced phosphorylation of the sodium channel, thus leading to a significant decrease in muscle excitability.

Effects of chronic sepsis on rat motor units: Experimental study of critical illness polyneuromyopathy

Critical illness polyneuromyopathy (CIP) leads to major muscle weakness correlated with peripheral nerve and/or muscle alterations. Because sepsis seems to be the main factor, we used an experimental model of chronic sepsis in rats to study the localization of the first alterations on isolated motor units of soleus muscle. Seven days of chronic sepsis leads to a decrease in muscle force and an increase in muscle fatigability. Muscle twitch contraction time is also slower and all the motor units exhibit a slow profile in septic rats. Motor axon conduction velocity remains normal. We observed a significant increase in the latency between nerve and muscle action potentials but no modifications in the electromechanical delay. The first action of sepsis on motor units seems to be a delayed trigger of muscle action potential along with a muscle weakness but without nerve conduction impairment.

Annexin A5 increases the cell surface expression and the chloride channel function of the ΔF508-cystic fibrosis transmembrane regulator

Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In CF, the most common mutant DeltaF508-CFTR is misfolded, is retained in the ER and is rapidly degraded. If conditions could allow DeltaF508-CFTR to reach and to stabilize in the plasma membrane, it could partially correct the CF defect. We have previously shown that annexin V (anxA5) binds to both the normal CFTR and the DeltaF508-CFTR in a Ca(2+)-dependent manner and that it regulates the chloride channel function of Wt-CFTR through its membrane integration. Our aim was to extend this finding to the DeltaF508-CFTR. Because some studies show that thapsigargin (Tg) increases the DeltaF508-CFTR apical expression and induces an increased [Ca(2+)](i) and because anxA5 relocates and binds to the plasma membrane in the presence of Ca(2+), we hypothesized that the Tg effect upon DeltaF508-CFTR function could involve anxA5. Our results show that raised anxA5 expression induces an augmented function of DeltaF508-CFTR due to its increased membrane localization. Furthermore, we show that the Tg effect involves anxA5. Therefore, we suggest that anxA5 is a potential therapeutic target in CF.

Effects of immobilizing a single muscle on the morphology and the activation of its muscle fibers

A single muscle of Wistar female rats, either soleus or peroneus longus, was immobilized by fixing its cut distal tendon to the bone during 8 weeks. We observed a transitory weight loss in both muscles; the mean fiber cross-sectional area (CSA) showed a reduction at day 30, followed by an increase at day 60. The time course of the activation of the immobilized muscle was evaluated by recording the chronic electromyographic (EMG) activity during short periods (1 min every other day) of treadmill locomotion. During immobilization, the integrated EMG amplitude of the soleus increased, reaching a maximum at 4 weeks, but remained close to control values during 8 weeks for the peroneus. The median frequency (MF) of the power density spectrum of the soleus EMG was not statistically different between immobilized and control muscles, while MF of the immobilized peroneus EMG was permanently higher than that of control muscles. This suggests two different modes of adaptation in motor unit command, depending on the muscle profile, which could be concomitant with the restoration of muscle fibers CSA after 8 weeks.

Characterization of the voltage-activated currents in cultured atrial myocytes isolated from the heart of the common oyster Crassostrea gigas.

SUMMARY Using the macro-patch clamp technique, we show that cardiac myocytes isolated from the heart of the oyster Crassostrea gigas possess several types of voltage-activated ionic currents. (1) A classical non-inactivating potassium current of the IK type that is inhibited by tetraethyl ammonium and shows an outward rectification and a slow activation. (2) A potassium current of the IA type that shows rapid activation and inactivation, and is blocked by 4-amino pyridine or preliminary depolarisation. (3) A potassium calcium-dependent current that is inhibited by charybdotoxin, activated by strong depolarisations and shows a large conductance. (4) A calcium inward current of the L-type that is inhibited by verapamil, cobalt and high concentrations of cadmium. This current is identified in most cells, but a T-type calcium current and classical fast sodium current are only identified in few cells, and only after a strong hyperpolarizing pulse. This suggests that these channels are normally inactivated in cultured cells and are not involved in the spontaneous activity of these cells. When they exist, the fast sodium channel is blocked by tetrodotoxin. The L-type calcium conductance is increased by serotonin. The identification in cultured oyster atrial cells of classical ionic currents, which are observed in most vertebrate species but only in a few species of molluscs, demonstrates that these cells are an interesting model. Moreover the viability and the electrophysiological properties of these cells are not significantly modified by freezing and thawing, thus increasing their usefulness in various bioassays.