Communication between motor nerves and skeletal muscle is dependent upon acetylcholine receptors (AChRs) expressed on the post-synaptic muscle cell membrane. As development proceeds the expression of these receptors is down-regulated by elevated muscle activity induced by the motor nerve. This down regulation occurs in all regions of the muscle cells with the exception of the nerve-muscle synapse. Associated with the muscle contractions are transient elevations in free cytoplasmic calcium and it is widely accepted that this elevation of intracellular calcium is responsible for the down-regulation of AChR expression. Yet it remains to be answered how AChR production can be maintained at an active nerve-muscle synapse if AChR production is suppressed in the rest of the cell. One possibility is that the nerve releases a factor which is capable of suppressing the effects of calcium in the region of the synapse. One such factor which has been suggested in the literature is the calcitonin gene-related peptide (CGRP). CGRP is present in motor nerve terminals and elevates cAMP levels is muscle cells in a G-Protein dependent manner. The Salpeter group has found that, in cultured rat muscle, the down-regulation of AChR expression caused by ryanodine (which alters sarcoplasmic reticulum calcium handling) or by the calcium ionophore A23187 is blocked by simultaneous treatment with the cAMP analogue dibutyryl cAMP. Thus it is possible that local elevation of cAMP at the nerve-muscle synapse could maintain AChR production in this region while elevated calcium in the rest of the muscle cells suppresses AChR production in the extrasynaptic region. The purpose of this study is to accurately measure cytoplasmic free calcium levels in mature cultured rat muscle cells after treatment with calcium mobilizers in the presence or absence of dibutyryl cAMP. The answers obtained from this study will enable us to determine if an intracellular cAMP signal, such as could be elicited by CGRP, causes changes in muscle cell calcium homeostasis or if the observed effect of cAMP analogues on AChR expression is due to regulation of an event down-stream of elevated calcium. Such information will be critical for elucidating the mechanism of AChR expression in mature synapses and will provide useful information to the study of synaptogenesis in general.
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