The expression of nicotinic acetylcholine receptors by PC12 cells treated with NGF.
The expression of neuronal nicotinic ACh receptors (nAChRs) and the subunits that compose these receptors by PC12 cells exposed to NGF has been studied. The analysis of total RNA reveals that the neuronal nAChR subunits alpha 3, alpha S, beta 2, beta 3, and beta 4, but not alpha 2 and alpha 4, are expressed in our PC12 cells. Within 48 hr of adding NGF to cultures, the RNA corresponding to alpha 3, alpha 5, beta 3, and beta 4 is decreased, but the beta 2 RNA increases for up to 6 d after NGF treatment. To determine the influence of NGF treatment on subunit protein expression, subunit-specific antisera were prepared. Immunocytochemistry detected antigen for alpha 3, alpha 5, beta 2, beta 3, and beta 4 (but not alpha 2 and alpha 4) in both NGF-treated and nontreated PC12 cells. The expression of nAChR subunit proteins, as measured by direct binding of antibodies to PC12 cells, does not change subsequent to 6 d of treatment with NGF. Whole-cell recording of PC12 cells shows that both the individual cell current density and response to the agonist cytisine were not altered after 5-7 d in NGF. However, the number of cells exhibiting detectable ACh-induced currents doubled. These results indicate that NGF increases the number of PC12 cells expressing ACh-sensitive nAChR currents but the activation is not the result of altering the amounts of individual nAChR subunit proteins. These data, taken together with the decrease in most nAChR subunit RNAs (except beta 2), suggest that NGF regulation of nAChRs may be through a posttranscriptional mechanism.
Rogers
S W
SW
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Mandelzys
A
A
Deneris
E S
ES
Cooper
E
E
Heinemann
S
S
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D016716:PC12 Cells; D012334:RNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000032:analysis / Q000096:biosynthesis* / Q000187:drug effects; D012260:Ribonucleases
D004273:DNA, Neoplasm; D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D012334:RNA, Neoplasm; D011978:Receptors, Nicotinic; D012260:Ribonucleases; D000109:Acetylcholine