Limits...
Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.

Nelson ME, Wang F, Kuryatov A, Choi CH, Gerzanich V, Lindstrom J - J. Gen. Physiol. (2001)

Bottom Line: Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells.Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells.The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1).

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA.

ABSTRACT
We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.

Show MeSH

Related in: MedlinePlus

Incubation in Nic increases the amount of α3β2 AChRs in IMR-32 and SH-SY5Y cells, but not α3β4 AChRs. The bar graph represents the relative amounts of [3H]epibatidine binding to AChRs immunoisolated on mAb-coated microwells from Triton-X100 extracts of either IMR-32 or SH-SY5Y cells in the presence and absence of Nic (100 μM) overnight. mAb 210 binds α3 and α5 AChRs, mAb 295 binds β2 AChRs, and mAb 337 binds β4 AChRs. For both cell lines, Nic caused an increase in binding to AChRs isolated on 210-coated wells which was the same in magnitude as the amount of increase in binding to AChRs isolated on mAb 295-coated wells. AChRs isolated on mAb 337-coated wells were not altered significantly by Nic. For IMR-32 (untreated), mAb 210 isolated AChRs bound ∼12 fmol of [3H]epibatidine, which corresponds to roughly 1,800 AChRs per cell when assuming two binding sites per AChR, whereas for SH-SY5Y, mAb 210 isolated AChRs bound ∼3 fmol of [3H]epibatidine corresponding to ∼600 AChRs per cell.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233843&req=5

Figure 10: Incubation in Nic increases the amount of α3β2 AChRs in IMR-32 and SH-SY5Y cells, but not α3β4 AChRs. The bar graph represents the relative amounts of [3H]epibatidine binding to AChRs immunoisolated on mAb-coated microwells from Triton-X100 extracts of either IMR-32 or SH-SY5Y cells in the presence and absence of Nic (100 μM) overnight. mAb 210 binds α3 and α5 AChRs, mAb 295 binds β2 AChRs, and mAb 337 binds β4 AChRs. For both cell lines, Nic caused an increase in binding to AChRs isolated on 210-coated wells which was the same in magnitude as the amount of increase in binding to AChRs isolated on mAb 295-coated wells. AChRs isolated on mAb 337-coated wells were not altered significantly by Nic. For IMR-32 (untreated), mAb 210 isolated AChRs bound ∼12 fmol of [3H]epibatidine, which corresponds to roughly 1,800 AChRs per cell when assuming two binding sites per AChR, whereas for SH-SY5Y, mAb 210 isolated AChRs bound ∼3 fmol of [3H]epibatidine corresponding to ∼600 AChRs per cell.

Mentions: For IMR-32 cell extracts, [3H]epibatidine binding revealed that 65 ± 5% (n = 5) of AChRs bound contained the β4 subunit when compared with those which contained the α3 subunit, whereas 18 ± 4% contained the β2 subunit (Fig. 10 and Table ). Additionally, the amount of AChR that could be labeled by [3H]epibatidine on intact IMR-32 cells (4.7 ± 0.8 fmol per 35-mm dish) was not significantly different from the amount of AChR that could immunoisolated from detergent extracts from IMR-32 cells (4.0 ± 0.1 fmol per 35-mm dish). Previously, we found that prolonged incubation of another neuroblastoma cell, SH-SY5Y, in Nic increased total α3 AChRs (Peng et al. 1997). Subsequently, we determined that Nic incubation increased the amount of AChR in α3β2-transfected cells, but had no effect on α3β4-transfected cells, and that in SH-SY5Y, α3β2, but not α3β4 AChRs were upregulated (Wang et al. 1998). With this in mind, we tested Nic incubation on IMR-32 cells. Overnight Nic (100 μM) treatment increased total α3-containing AChRs by ∼40% (Fig. 10 and Table ), most of which could be attributed to an increase in β2-containing AChRs. Similar to previous findings in SH-SY5Y cells, Nic incubation increased the amount of α3 AChRs by ∼80%, most of which could be attributed to an increase in β2-containing AChRs.


Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.

Nelson ME, Wang F, Kuryatov A, Choi CH, Gerzanich V, Lindstrom J - J. Gen. Physiol. (2001)

Incubation in Nic increases the amount of α3β2 AChRs in IMR-32 and SH-SY5Y cells, but not α3β4 AChRs. The bar graph represents the relative amounts of [3H]epibatidine binding to AChRs immunoisolated on mAb-coated microwells from Triton-X100 extracts of either IMR-32 or SH-SY5Y cells in the presence and absence of Nic (100 μM) overnight. mAb 210 binds α3 and α5 AChRs, mAb 295 binds β2 AChRs, and mAb 337 binds β4 AChRs. For both cell lines, Nic caused an increase in binding to AChRs isolated on 210-coated wells which was the same in magnitude as the amount of increase in binding to AChRs isolated on mAb 295-coated wells. AChRs isolated on mAb 337-coated wells were not altered significantly by Nic. For IMR-32 (untreated), mAb 210 isolated AChRs bound ∼12 fmol of [3H]epibatidine, which corresponds to roughly 1,800 AChRs per cell when assuming two binding sites per AChR, whereas for SH-SY5Y, mAb 210 isolated AChRs bound ∼3 fmol of [3H]epibatidine corresponding to ∼600 AChRs per cell.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233843&req=5

Figure 10: Incubation in Nic increases the amount of α3β2 AChRs in IMR-32 and SH-SY5Y cells, but not α3β4 AChRs. The bar graph represents the relative amounts of [3H]epibatidine binding to AChRs immunoisolated on mAb-coated microwells from Triton-X100 extracts of either IMR-32 or SH-SY5Y cells in the presence and absence of Nic (100 μM) overnight. mAb 210 binds α3 and α5 AChRs, mAb 295 binds β2 AChRs, and mAb 337 binds β4 AChRs. For both cell lines, Nic caused an increase in binding to AChRs isolated on 210-coated wells which was the same in magnitude as the amount of increase in binding to AChRs isolated on mAb 295-coated wells. AChRs isolated on mAb 337-coated wells were not altered significantly by Nic. For IMR-32 (untreated), mAb 210 isolated AChRs bound ∼12 fmol of [3H]epibatidine, which corresponds to roughly 1,800 AChRs per cell when assuming two binding sites per AChR, whereas for SH-SY5Y, mAb 210 isolated AChRs bound ∼3 fmol of [3H]epibatidine corresponding to ∼600 AChRs per cell.
Mentions: For IMR-32 cell extracts, [3H]epibatidine binding revealed that 65 ± 5% (n = 5) of AChRs bound contained the β4 subunit when compared with those which contained the α3 subunit, whereas 18 ± 4% contained the β2 subunit (Fig. 10 and Table ). Additionally, the amount of AChR that could be labeled by [3H]epibatidine on intact IMR-32 cells (4.7 ± 0.8 fmol per 35-mm dish) was not significantly different from the amount of AChR that could immunoisolated from detergent extracts from IMR-32 cells (4.0 ± 0.1 fmol per 35-mm dish). Previously, we found that prolonged incubation of another neuroblastoma cell, SH-SY5Y, in Nic increased total α3 AChRs (Peng et al. 1997). Subsequently, we determined that Nic incubation increased the amount of AChR in α3β2-transfected cells, but had no effect on α3β4-transfected cells, and that in SH-SY5Y, α3β2, but not α3β4 AChRs were upregulated (Wang et al. 1998). With this in mind, we tested Nic incubation on IMR-32 cells. Overnight Nic (100 μM) treatment increased total α3-containing AChRs by ∼40% (Fig. 10 and Table ), most of which could be attributed to an increase in β2-containing AChRs. Similar to previous findings in SH-SY5Y cells, Nic incubation increased the amount of α3 AChRs by ∼80%, most of which could be attributed to an increase in β2-containing AChRs.

Bottom Line: Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells.Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells.The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1).

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA.

ABSTRACT
We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human alpha3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express alpha3, alpha5, alpha7, beta2, and beta4 AChR subunits. From these subunits, several types of alpha3 AChRs as well as homomeric alpha7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect alpha3beta4 AChRs. alpha7 AChR function was not detected, yet we estimate that there are 70% as many surface alpha7 AChRs in IMR-32 when compared with alpha3 AChRs. Agonist potencies (EC(50) values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16+/-1 microM) > nicotine (Nic; 48 +/- 7 microM) > or = cytisine (Cyt; 57 +/- 3 microM) = acetylcholine (ACh; 59 +/- 6 microM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s(-1) (300 microM ACh; -60 mV). Assays that used mAbs confirmed the predominance of alpha3- and beta4-containing AChRs in IMR-32 cells. Although 18% of total alpha3 AChRs contained beta2 subunits, no beta2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface alpha3beta2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in alpha3beta2 transfected HEK cells. Characterization of various alpha3 AChRs expressed in HEK cell lines revealed that the functional properties of the alpha3beta4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC(50) values) for this line was DMPP (14 +/- 1 microM) = Cyt (18 +/- 1 microM) > Nic (56 +/- 15 microM > ACh (79 +/- 8 microM). The efficacies of both Cyt and DMPP were approximately 80% when compared with ACh and the desensitization rate was 2 s(-1). These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to alpha3beta4 AChRs.

Show MeSH
Related in: MedlinePlus