Muscarinic receptor subtypes in human neuroblastoma cell lines SH-SY5Y and IMR-32 as determined by receptor binding, Ca++ mobilization and northern blotting.
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.
Kukkonen
J
J
Department of Biochemistry and Pharmacy, Abo Akademi University, Biocity, Turku, Finland.
Ojala
P
P
Näsman
J
J
Hämäläinen
H
H
Heikkilä
J
J
Akerman
K E
KE
D015152:Blotting, Northern; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D019832:N-Methylscopolamine; D009447:Neuroblastoma / Q000737:chemistry*; D010716:Phosphatidylinositols / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology; D010890:Pirenzepine / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000032:analysis* / Q000235:genetics; D012602:Scopolamine Derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D010716:Phosphatidylinositols; D010880:Piperidines; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D010890:Pirenzepine; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D002118:Calcium; D019832:N-Methylscopolamine