Katsuya Dezaki

Immunohistochemical localization of neuronal nicotinic receptor subtypes at the pre-and postjunctional sites in mouse diaphragm muscle

The existence of neuronal nicotinic acetylcholine receptor (nAChR) subunits was investigated in the cryostat sections of mouse diaphragm muscles using the indirect immunofluorescence technique. The specific immunolabelings with monoclonal antibodies (mAbs) to beta 2 and to alpha 8 subunits of neuronal nAChR were observed at the endplate determined by labeling with a fluorescent dye (BODIPY)-conjugated alpha-bungarotoxin. The immunoreactivity of mAb to the alpha 3 subunit of neuronal nAChR was detected on the motor nerve fibers including the nerve terminals. These results provide evidence that the subtypes of postsynaptic nAChR, recognized by the anti-beta 2 and/or anti-alpha 8 mAbs, and the presynaptic nAChR recognized by the anti-alpha 3 mAb, are present at the neuromuscular junction, in addition to the classical muscle nAChR.

Enhancement by calcitonin gene‐related peptide of nicotinic receptor‐operated noncontractile Ca2+ mobilization at the mouse neuromuscular junction

1 The involvement of calcitonin gene‐related peptide (CGRP) in the mechanism of nicotinic acetylcholine receptor‐operated noncontractile Ca2+ mobilization (not accompanied by twitch tension) was investigated by measuring Ca2+‐aequorin luminescence at the neuromuscular junction of mouse diaphragm muscle treated with neostigmine. 2 Noncontractile Ca2+ transients were enhanced by 4‐aminopyridine (100 μm), a K+ channel blocker, and inhibited by botulinum toxin (1–100 μg, i.p.) and hexamethonium (10–100 μm), a neuronal nicotinic receptor antagonist. 3 Noncontractile Ca2+ transients were diminished by CGRP8–37 (10–20 μm), a CGRP antagonist. CGRP (0.3–10 nm) prolonged the duration of noncontractile Ca2+ transients. The effect of CGRP was suppressed by CGRP8_37 (0.1 μm). 4 Noncontractile Ca2+ transients were inhibited by H‐89 (0.1–1 μm), a protein kinase‐A inhibitor. The catalytic subunit of protein kinase‐A and AA373 (300 μm), a protein kinase‐A activator, prolonged the duration of noncontractile transients. The prolongations either by CGRP or by AA373 were not observed in the presence of H‐89 (0.1 μm). 5 Contractile (accompanied by twitch tension) but not noncontractile Ca2+ transients were decreased by 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA, 0.3–1 μm), a protein kinase‐C activator. Phospholipase A2 increased only contractile Ca2+ transients. Calmodulin‐related agents affected neither type of Ca2+ transients. 6 These results provide the first evidence that nicotinic acetylcholine receptor‐operated noncontractile Ca2+ mobilization is promoted by nerve‐released CGRP activating protein kinase‐A, and is dependent on the accumulated amounts of acetylcholine at the neuromuscular junction where desensitization might readily develop.

Postsynaptic nicotinic receptor desensitized by non-contractile Ca2+ mobilization via protein kinase-C activation at the mouse neuromuscular junction

1 Non‐contractile Ca2+ mobilization (unaccompanied by muscle contraction) was initiated by nerve stimulation in the presence of neostigmine (more than 0.03 μm) at the endplate region of mouse diaphragm muscles. In the process of nicotinic receptor desensitization, the depressant effect of non‐contractile Ca2+ on contractile Ca2+ mobilization was investigated by measurement of Ca2+‐aequorin luminescence. 2 When the phrenic nerve was stimulated with paired pulses having intervals of 150, 300, 600, 1000 and 2000 ms, contractile Ca2+ transients were elicited during the generation of non‐contractile Ca2+ mobilization. The amplitude of the contractile Ca2+ transients elicited by the second pulse (S2) was depressed at the shorter pulse intervals, but recovered to the initial contractile response (S1) at longer pulse intervals. 3 The extent of depression of S2 was enhanced by increasing the concentration of neostigmine (0.03 to 0.3 μm). When a low concentration (0.05 μm) of pancuronium, a competitive nicotinic antagonist, completely blocked non‐contractile Ca2+ mobilization, the depression of S2 was diminished. 4 The depression of S2 was enhanced when the peak amplitude of non‐contractile Ca2+ mobilization was raised by increasing the external Ca2+ concentration from 1.3 to 5 mm. 5 Staurosporine (10 nm), a protein kinase‐C inhibitor, diminished the depression of S2 despite large amounts of non‐contractile Ca2+ mobilization. The diminishing effect of staurosporine was counteracted by TPA (0.1 μm), a protein kinase‐C activator. 6 These findings suggest that non‐contractile Ca2+ mobilization may enhance the desensitization of the postsynaptic nicotinic receptor via activation of protein kinase‐C at the neuromuscular junction.

Enhancement by calcitonin gene‐related peptide of non‐contractile Ca2+‐induced nicotinic receptor desensitization at the mouse neuromuscular junction

1 Nicotinic acetylcholine receptor (AChR)‐operated non‐contractile Ca2+ mobilization (unaccompanied by muscle contraction) depressed contractile Ca2+ mobilization (accompanied by muscle contraction) in mouse diaphragm muscles. In the process of nicotinic AChR desensitization, the enhancing role of calcitonin gene‐related peptide (CGRP) on the non‐contractile Ca2+‐induced depression of contractile Ca2+ mobilization was investigated by measurement of Ca2+‐aequorin luminescence in the presence of neostigmine (0.1 μm). 2 When the phrenic nerve was stimulated with paired pulses at intervals of 150, 300, 600, 1000 and 2000 ms, contractile Ca2+ transients were elicited during the generation of non‐contractile Ca2+ mobilization. The amplitude of the contractile Ca2+ transients elicited by the second pulse (S2) was depressed at the shorter pulse intervals, but not at the longer pulse intervals. 3 The extent of depression of S2 was enhanced when the duration of non‐contractile Ca2+ mobilization was prolonged by CGRP (10 nM). However, CGRP failed to enhance the depression of S2 when non‐contractile Ca2+ mobilization was not observed at the low external Ca2+ concentration (1.3 mM). 4 The enhancing effect by CGRP on the depression of S2 was counteracted by staurosporine (3 nM), a protein kinase‐C inhibitor, despite prolongation of the duration of non‐contractile Ca2+ mobilization. 5 When H‐89 (1 μm), a protein kinase‐A inhibitor, completely blocked non‐contractile Ca2+ mobilization, the depression of S2 was diminished. The prolongation of the duration of non‐contractile Ca2+ mobilization by AA373 (300 μm), a protein kinase‐A activator, enhanced the depression of S2. The enhancing effect was observed neither with CGRP nor with AA373, in the presence of H‐89 (0.1 μm). 6 These findings suggest that the CGRP mobilizes non‐contractile Ca2+ through activation of protein kinase‐A, which in turn may activate protein kinase‐C, then enhance the desensitization of postsynaptic nicotinic AChRs at the neuromuscular junction.

Monoclonal antibody to β2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca2+ mobilization in the mouse diaphragm muscle

The involvement of subtypes of nicotinic acetylcholine receptor (nAChR) in the postjunctional non-contractile Ca2+ mobilization was investigated in mouse diaphragm muscles treated with an anticholinesterase, using monoclonal antibodies (mAbs) to nAChR subunits. mAb 210 (specific for alpha 1 subunit of muscle nAChR) depressed contractile Ca2+ transients without affecting non-contractile Ca2+ transients. mAb 270 (specific for beta 2 subunit of neuronal nAChR) depressed only non-contractile Ca2+ transients. mAb 210 did not completely block the ACh-activated channel currents in flexor digitorum brevis muscle cells. The present findings indicate that the anti-beta 2 mAb 270-related subtype of nAChR may postsynaptically operate the non-contractile Ca2+ mobilization at the neuromuscular junction, suggesting the involvement of a subtype different from the usual muscle-type nAChR.

Calcitonin gene-related peptide potentiates nicotinic acetylcholine receptor-operated slow Ca2+ mobilization at mouse muscle endplates.

1 The involvement of calcitonin gene‐related peptide (CGRP) in the non‐contractile slow Ca2+ mobilization induced by prolonged nicotinic stimulation was investigated by measurement of [Ca2+]i levels in mouse single muscle cells (flexor digitorum brevis; FDB) loaded with a Ca2+ indicator fluo‐3 using confocal laser scanning microscopy. 2 CGRP (3–30 nM) potentiated acetylcholine (ACh, 1 μM)‐elicited slow Ca2+ mobilization in a concentration‐dependent manner. 3 The potentiation by CGRP of the slow Ca2+ component was greatly depressed by a competitive nicotinic antagonist (+)‐tubocurarine (5 μM). The Ca2+ channel blocker nitrendipine (1 μM) affected neither ACh responses nor the CGRP potentiation. 4 The slow Ca2+ component was completely abolished by reducing [Ca2+]0 from 2.5 to 0.25 mM whereas the fast component was not affected. The CGRP‐induced potentiation of slow Ca2+ signal was also depressed by decreasing [Ca2+]0. 5 Isoproterenol (30 μM) and 8‐bromo‐adenosine 3′,5′‐cyclic monophosphate (1 mM) potentiated the ACh‐elicited slow Ca2+ response. The potentiation by CGRP of the slow Ca2+ component was completely abolished by a protein kinase‐A inhibitor H‐89 (1 μM). 6 These findings indicate that CGRP potentiates the nicotinic ACh receptor‐operated slow Ca2+ signal via the activation of protein kinase‐A system at the skeletal muscle endplates.

Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate.

Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve‐diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+‐aequorin luminescence. ACh (0.1–3 mm, 20 μl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration‐dependently increased by ACh, whereas that of the fast component (accompanied by twitch tension) reached a maximum response at a lower concentration (0.1 mm) of applied ACh. A pulse of nicotinic agonists, (−)‐nicotine (10 mm) and 1,1‐dimethyl‐4‐phenyl‐piperazinium (10 mm), but not a muscarinic agonist pilocarpine (10 mm), also elicited a biphasic Ca2+ signal. Even though ACh release from motor nerve endings was blocked by botulinum toxin (5 μg, bolus i.p. before isolation of the tissue), the generation of both a fast and slow Ca2+ component caused by ACh application was observed. These results strongly suggest that ACh locally applied onto the endplate region of skeletal muscle induces a slow Ca2+ signal reflecting Ca2+ entry through a postsynaptic nicotinic AChR, which has a low sensitivity to transmitter ACh.

Neuronal nicotinic receptor operates slow Ca2+ mobilization at mouse muscle endplate

The contribution to neuromuscular functions by neuronal nicotinic acetylcholine receptor (nAChR) expressed at skeletal muscle endplate was investigated using intracellular Ca2+ measurements. A neuronal nAChR blocker, methyllycaconitine (MLA), depressed non-contractile Ca2+ mobilization without affecting muscle nAChR activity in nerve-stimulated mouse diaphragm muscle, after cholinesterase inhibition. Confocal imaging demonstrates that the MLA-sensitive Ca2+ mobilization also occurred at the endplate in single flexor digitorum brevis muscle cells as the slow component of two-phasic Ca2+ elevation after the prolonged nicotinic stimulation. A monoclonal antibody to alpha 1 subunit of muscle nAChR depressed the fast but not the slow component. Thus, muscle neuronal-nAChR can induce the localized rise of Ca2+ at the postjunctional sites.

Methyllycaconitine-sensitive neuronal nicotinic receptor-operated slow Ca2+ signal by local application or perfusion of ACh at the mouse neuromuscular junction

Local application of acetylcholine (ACh; 0.3 mM, 20 microl) elicited bi-phasic elevation of intracellular Ca2+ concentrations (contractile fast and non-contractile slow Ca2- signal measured as aequorin luminescence) in diaphragm muscle preparation. A neuronal nicotinic antagonist methyllycaconitine (MLA; 0.01-1 microM), which did not affect the fast Ca2+ transients and twitch tension, concentration-dependently depressed only the slow Ca2+ component. Ca2+ channel blockers, Cd2+ (200 microM), nitrendipine (1 microM), verapamil (1 microM) and diltiazem (1 microM), or a Na+ channel blocker tetrodotoxin (TTX; 0.1 microM) failed to prevent the generation of slow Ca2+ response. Perfusion of ACh (1 microM) to isolated single skeletal (flexor digitorum brevis) muscle cells pretreated with TTX (0.1 microM) also elicited a slow Ca2+ signal measured as confocal imaging with a fluorescent dye, fluo-3, at the endplate region. MLA (1 microM) antagonized against the ACh perfusion-elicited slow Ca2+ signal. Perfusion of choline (1 mM), a neuronal nicotinic agonist, also elicited the MLA-sensitive slow Ca2+ signal. These results strongly suggest that the ACh-induced slow Ca2+ signal reflects Ca2+ entry through a postsynaptic MLA-sensitive neuronal nicotinic ACh receptor subtype at the neuromuscular junction.

Diabetic state-induced rapid inactivation of noncontractile Ca2+ mobilization operated by nicotinic acetylcholine receptor in mouse diaphragm muscle.

1 Diabetic modifications of nicotinic receptor‐operated noncontractile Ca2+ mobilization observed in the presence of anticholinesterase were investigated by measuring Ca2+‐aequorin luminescence in diaphragm muscles of mice with diabetes induced by injections of streptozotocin (150 mg kg−1, bolus i.v.) and alloxan (85 mg kg−1, bolus i.v.). 2 The diabetic state accelerated the decline of noncontractile Ca2+ transients without affecting their peak amplitude. Insulin treatment reversed this alteration. 3 The increase in contractile Ca2+ transients by cholinesterase inhibition was attenuated 0.6 fold and became resistant to changes in [Ca2+]o in the diabetic state. 4 Changes in extracellular pH from 7.6 to 5.6 depressed the peak amplitude of noncontractile Ca2+ transients without affecting their duration, and enhanced the peak amplitude of contractile Ca2+ transients. 5 These results suggest that the inactivation process of noncontractile Ca2+ mobilization is promoted in diabetic muscles, presumably by desensitization of the nicotinic acetylcholine receptor.