Regulation of muscarinic receptor expression was examined in culture sympathetic neurons of the neonatal rat superior cervical ganglion. Receptor concentration was determined by measuring binding of the muscarinic antagonist 1-quinuclidinyl [phenyl-4-3H]benzilate (3H-QNB). 3H-QNB bound to one apparent class of noninteracting sites on sympathetic neuron membranes with a K(d) of 28.9 pM and a B(max) of 2.91 pmol/mg protein. Binding increased as a linear function of tissue protein and was saturable. The number of receptors per milligram protein increased approximately 6-fold during 16 d of culture, and receptor numbers were down-regulated by treatment with the agonist carbachol. These observations suggested that measurement of 3H-QNB binding would provide a reliable estimate of muscarinic receptor number on cultured sympathetic neurons. To determine whether non-neuronal cells produce soluble factors that influence muscarinic receptor expression, the effects of treatment with rat fibroblast-conditioned medium (RFCM) were examined. Exposure of sympathetic neurons to 50% RFCM resulted in a 57% decrease in muscarinic receptor numbers without a change in the apparent K(d). The decrease in 3H-QNB binding in response to RFCM was dose-related, with a minimum dose of 15% RFCM required to observe a significant effect. In contrast to the carbachol-induced down-regulation, the reduction in binding after RFCM treatment was not prevented by atropine, indicating that the effect was not mediated by acetylcholine stimulation of muscarinic receptors. Binding of 125I-alpha-bungarotoxin, which labels a nonfunctional membrane site distinct from nicotinic receptors, was not altered by treatment with RFCM, indicating the selectivity of the change in membrane muscarinic receptors. Moreover, RFCM treatment increased levels of the cholinergic biosynthetic enzyme, choline acetyltransferase. These observations suggest that the complement of receptors expressed by sympathetic neurons may be regulated by soluble factors produced by non-neuronal cells, and that transmitter and receptor phenotypes may both be regulated by factors produced by a single type of non-neuronal cell.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Neuroscience|
|State||Published - 1988|
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