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MC Steel and NJ Buckley
National Institute for Medical Research, London, UK.
The human neuroblastoma line SH-SY5Y expresses three muscarinic receptor genes (m1, m2, and m3). In this study, we have investigated the effect of agonist exposure on the steady state levels of each muscarinic receptor transcript, using a comparative polymerase chain reaction (PCR) assay that allows changes in levels of very rare transcripts to be monitored. Northern blot analysis of cellular RNA revealed the presence of m3 mRNA, whereas PCR amplification of SH-SY5Y cDNA additionally revealed the presence of m1 and m2 transcripts. Cell surface muscarinic receptor number, as assessed by N- [3H]methylscopolamine binding to whole cells, rapidly decreased to 42% of control levels within 1 hr of exposure to 100 microM carbachol; this was followed by a slower decline to 6% of control levels after 48 hr. Total receptor number, measured by binding of [3H]quinuclidinyl benzilate, showed a much slower decline to 21% of control levels after 48 hr of treatment. Comparative PCR analysis showed that each muscarinic transcript was differentially regulated. The level of transcript encoding the major receptor population, the m3 mRNA, was rapidly elevated within 1 hr of agonist challenge and subsequently decreased to about 30% of prestimulation levels within 9 hr; this decrease was sustained for the time course of the experiment. m2 mRNA levels showed a transient increase followed by a decrease to 30% of prestimulation levels after 6 hr but, in contrast to the m3 transcripts, this depression was followed by a transient rise to 270% of prestimulation levels after 24 hr before declining to normal levels by 72 hr after stimulation. Exposure of cells to agonist clearly instigates a complex pattern of changes in levels of receptor and receptor mRNA; comparison of the relative time courses of these changes indicates that the decline in m3 transcripts precedes the loss of muscarinic receptor binding sites.
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