Enhancement by calcitonin gene-related peptide of noncontractile 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 S, when noncontractile 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.