Thelma C. Madhok

Nicotine regulates nicotinic cholinergic receptors and subunit rnRNAs in PC 12 cells through protein kinase A

To understand the up-regulation of neuronal nicotinic cholinergic receptors (nAcChRs) that results from chronic in vivo treatment with nicotine, we studied the effect of nicotine on [3H]nicotine binding sites on PC 12 cells. PC 12 cells were grown in nicotine hemisulfate (10(-6) to 10(-3) M) or vehicle for 7 days, and specific [3H]nicotine binding was measured. Nicotine (10(-6) to 10(-4) M) dose-dependently increased specific binding by up to 2.6-fold over basal levels in 5-7 days, whereas a 10(-3) M concentration failed to do so. In contrast, [3H]nicotine binding to PC 12 cell mutants (A126.1B2 and A123.7), deficient in cAMP-responsive protein kinase A Types I and/or II, was unaffected by nicotine. Northern gel analysis of nAcChR subunit mRNAs from wild type PC 12 cells showed that the mRNA encoding the dominant agonist-binding subunit, alpha 3, was significantly reduced by nicotine, as early as 4 h after treatment, whereas mRNA for the structural beta 2 subunit was slightly increased. In contrast, the alpha 3 subunit mRNA from the PC 12 cell mutant A123.7 was not significantly decreased after 4 h and 7 days of nicotine treatment. These studies indicate that nicotine up-regulates expression of nAcChRs on wild type PC 12 cells and reduces the content of alpha 3 subunit mRNA; these effects require an intact protein kinase A system. The divergent effects of nicotine on the nAcChR compared to its alpha 3 subunit mRNA suggests that enhanced expression of nicotinic receptors may not involve synthesis of new receptor subunit proteins.

Characterization of uterine estrogen receptors by size-exclusion and ion-exchange high-performance liquid chromatography.

This report presents the first application of ion-exchange high-performance liquid chromatography in the study of ER from the rabbit uterus. In the presence of sodium molybdate (20 mM), native ER was eluted as a sharp peak at 0.29 M NaCl by a linear salt gradient, but without molybdate, it resolved into 4 major peaks. Molybdate-stabilized ER from the DEAE column, similar to ER from crude cytosol, sedimented at the 6-8S region in low salt and 4S region in high salt linear sucrose gradients, and was excluded from size-exclusion HPLC. In contrast, dissociated ER subunits from DEAE eluates ranged from 3.5 to 4.5S, and showed differences in molecular weights in a size-exclusion column. These results show that the native ER is a large molecule which dissociates into smaller subunits in the absence of molybdate; each of the steroid-bound moieties differs in molecular weight and surface charge from the native molecule.

Monospecific antibodies against a synthetic peptide predicted from the alpha-3 nicotinic receptor cDNA inhibit binding of [3H]nicotine to rat brain nicotinic cholinergic receptor.

Polyclonal antibodies were raised against a synthetic decapeptide (designated S3) predicted from a segment of the alpha-3 subunit cDNA (amino acid residues 130-139) encoding the rat brain nicotinic cholinergic receptor. This segment was selected because it may be proximate to the nicotine/acetylcholine-binding site of the receptor (1). By radioligand binding assays and sucrose density gradient centrifugation, these monospecific antibodies were shown to inhibit the binding of [3H]nicotine to both the large molecular weight rat brain receptor (240 kDa) and to an SDS-disaggregated nicotine-binding subunit species (80 kDa), in a dose-dependent manner. The neutralizing effect of the anti-S3 antibodies supports the view that this region of the protein is closely related to the agonist binding site.

Characterization of Molybdate-Stabilized Estrogen Receptors by Hydrophobic Interaction HPLC: Resolution of Two 8S Subunits

This report describes the separation and characterization of estrogen receptors (ER) according to their degree of hydrophobicity and surface charges. Molybdate-stabilized [3H]ER from rabbit uterine cytosol was sequentially purified by passage through a size-exclusion pre-column, an anion-exchange column, and a hydrophobic interaction column. With fresh cytosol, a major radioactive peak was eluted from the DEAE columns; a major peak and a minor, less hydrophobic, peak were eluted from the hydrophobic column. In contrast, ER from frozen cytosol showed one peak in the DEAE-column and exhibited four radioactive peaks in the hydrophobic column. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, [3H] tamoxifen aziridine(TA)-labelled ER showed radioactive bands at 62 and 48 kd. The subunits which were characterized by these radioactive bands were successfully separated by the hydrophobic column; the more hydrophobic subunit corresponded to the 62 kd band. The HPLC-purified [3H]TA-labelled ER subunits sediment at a 7.4-8.5S region in a low-salt sucrose gradient. These results show that differential negative surface charge and hydrophobic areas exist in the holo-receptor and its subunits, and the hydrophobic interaction HPLC column separates the two major 8S steroid binding subunits of ER.

Brain nicotinic receptors isolated by a monospecific antibody against a synthetic α-3 subunit receptor peptide compared to a monoclonal anti-idiotypic (to nicotine) antibody

Nicotinic cholinergic receptor proteins purified from rat brain by immunoaffinity chromatography were characterized using the anti-S3 polyclonal antibody vs. the anti-idiotypic monoclonal antibody 422F11 (generated against an antibody to nicotine). Anti-S3 IgG was purified to homogeneity; anti-S3-Sepharose 4B and 422F11-Sepharose 4B each depleted 3H-nicotine binding sites from brain. Nicotinic receptors isolated from both immunoaffinity columns showed major bands (silver-stained) at 55K and 70K. Using anti-S3 serum as probe, Western blots of nicotinic receptors isolated by the two immunoaffinity gels also showed major bands at 55 and 70K. However, Western blots of fresh brain extracts revealed a major band at 80K and minor bands at 55K and 70K. These results show similar nicotinic cholinergic receptor proteins isolated by the anti-S3 and 422F11 anti-idiotypic antibodies; 80K was dominant only when fresh brain extract was subjected to Western blotting without prior immunoaffinity purification.

Purification of rabbit uterine cytosolic estrogen receptors by affinity chromatography

Abstract Progress to elucidate the mechanism of estrogen action in uterine tissues has been hampered because of the formidable task of receptor purification. This report describes our recent success in the purification of ERc from estrogen-primed rabbit uterus. Crude cytosolic ERc are purified sequentially by affinity chromatography and gel filtration, using heparin-Sepharose 4B, 17β-estradiol-17-hemisuccinyl-oval-bumin-Sepharose 4B, and Sephadex G-50. This purified ERc sediments as a 4.5 S molecule on sucrose gradients in low-salt buffer, and exhibits a major radioactive peak in both polyacrylamide gel electrophoresis under nondenaturing conditions and in HPLC eluates. The radioactivity pattern in HPLC coincides with that of the u.v. scan, showing the elimination of other u.v. absorbing materials of the crude cytosol. These results show that the bulk of cytosolic proteins have been removed by this procedure and indicate the feasibility of using HPLC for the large scale purification and analysis of ERc. Attempts are initiated in this laboratory to produce antibodies against this purified receptor by hybridoma technique, and to further analyze the biochemical nature of the purified receptor.

Analysis of Rat Uterine Estrogen Receptors by High-Pressure Liquid Chromatography Methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.