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Muscarinic receptor subtypes in human neuroblastoma cell lines SH-SY5Y and IMR-32 as determined by receptor binding, Ca++ mobilization and northern blotting

J Kukkonen, P Ojala, J Nasman, H Hamalainen, J Heikkila and KE Akerman

Department of Biochemistry and Pharmacy, Abo Akademi University, Biocity, Turku, Finland.

Muscarinic receptor subtypes in neuroblastoma cell lines IMR-32 and SH- SY5Y were determined with receptor binding, Ca++ mobilization and Northern blotting. Displacement of [3H]NMS with pirenzepine in IMR-32 cells revealed apparent binding sites with Kd values of 5 (41%) and 237 nM (59%). With 4-diphenylacetoxy-N-metylpiperidine metiodid, a similar proportion of apparent high- and low-affinity binding was obtained: 36 (Kd = 0.26 nM) and 64% (Kd = 6.3 nM), respectively. In SH-SY5Y cells, two different affinities with apparent Kd of 40 (24%) and 460 nM (76%) could be distinguished with pirenzepine, even though the Kd of the apparent high-affinity site varied markedly (variation = 8.7-96.8 nM). Inhibition of carbachol-induced Ca++ mobilization displayed high sensitivity to 4-diphenylacetoxy-N-methylpiperidine metiodid in both cell lines. IMR-32 cells displayed high sensitivity to pirenzepine, whereas the sensitivity varied between different batches of SH-SY5Y cells. DNA fragments (approximately 1000 base pairs) from SH-SY5Y DNA amplified with polymerase chain reaction were used as probes for muscarinic receptor mRNA. Northern blotting with the Hm1-specific probe gave a stronger signal for SH-SY5Y than for IMR-32, whereas the result obtained with the Hm2-probe was the opposite. Also, the Hm3 mRNA was detected in SH-SY5Y cells. The Hm4 and Hm5 transcripts were not detected in either of these cell lines.

Volume 263, Issue 3, pp. 1487-1493, 12/01/1992
Copyright © 1992 by American Society for Pharmacology and Experimental Therapeutics




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Copyright © 1992 by the American Society for Pharmacology and Experimental Therapeutics.