Non-neuronal cell-conditioned medium regulates muscarinic receptor expression in cultured sympathetic neurons

Abstract

Regulation of muscarinic receptor expression was examined in cultured 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 Kd of 28.9 pM and a Bmax 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 nonneuronal 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 Kd. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmentally Regulated Secreted Factors Control Expression of Muscarinic Receptor Subtypes in Embryonic Chick Retina

“…The effects of CM on expression of mAChR observed in this study are consistent with several other reports which showed that soluble factors can alter expression of mAChR, by either decreasing or increasing the levels of mAChR, or by increasing the number of cells that express mAChR. Fibroblast-conditioned media causes mAChR levels to decrease in rat sympathetic cultures (Smith and Kessler, 1988). The level of mAChR in cultured chick heart increases with treatment with brain extract (Siege1 and Fisch bach, 1984), and the number of mAChR-positive cells increases in rat enteric neurons cultured with heart-conditioned media (Nishi and Willard, 1988).…”

Localization, regulation and functions of neurotransmitters and neuromodulators in cervical sympathetic ganglia

“…On the other hand, LDVs are randomly distributed in the nerve terminal supporting the hypothesis of parasynaptic peptide release that requires high frequency stimulation (Lundberg et al, 1989) and takes place in a significantly slower time course with different Ca channels possibly involved (Troger et al, 1994). Whereas acetylcholine binds to ion channels at the site of nicotinic receptors (fast excitatory), the muscarinic cholinoceptors (slow excitatory; Ludlam and Smith and Kessler, 1988) as well as adrenoceptors and neuropeptide-binding sites represent guanine nucleotide-binding protein (G protein)-linked receptors consisting of seven membrane-spanning hydrophobic sequences (Millhorn et al, 1989). The target for NO is a cytoplasmic guanylate cyclase (Vincent, 1994).…”

Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation

“…Differentiation alone has been suggested to regulate the expression of P,-and &adrenergic receptor mRNA and protein in 3T3-Ll cells, a cell line that exhibits a fibroblast-like phenotype and can acquire an adipocyte-like phenotype (Guest et al, 1990). Conversely, both soluble factors and cell-cell interactions have been shown to influence the expression of muscarinic and P-adrenergic receptors in vitro (Harden et al, 1979;Smith and Kessler, 1988; reviewed in Grant and Landis, 199 1). It is not known, however, whether these mechanisms play a role Our studies have shown there is a close correlation between the level of m3 mRNA expression and the density of muscarinic binding sites throughout development.…”