Limits...
Characterization of ST14A Cells for Studying Modulation of Voltage-Gated Calcium Channels.

Roberts-Crowley ML, Rittenhouse AR - PLoS ONE (2015)

Bottom Line: Transfection with LTC subunits produced functional Ca(v)1.3 current from round cells, providing a homogeneous model system compared to native MSNs for studying D(2)R pathways.However, neither endogenous nor recombinant Ca(v)1.3 current was modulated by the D(2)R agonist quinpirole.Thus, ST14A cells provide a MSN-like cell line for studying channel modulation and signaling pathways that do not involve activation of PLCβ-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
In medium spiny neurons (MSNs) of the striatum, dopamine D2 receptors (D2Rs) specifically inhibit the Ca(v)1.3 subtype of L-type Ca(2+) channels (LTCs). MSNs are heterogeneous in their expression of dopamine receptors making the study of D2R pathways difficult in primary neurons. Here, we employed the ST14A cell line, derived from embryonic striatum and characterized to have properties of MSNs, to study Ca(v)1.3 current and its modulation by neurotransmitters. Round, undifferentiated ST14A cells exhibited little to no endogenous Ca(2+) current while differentiated ST14A cells expressed endogenous Ca(2+) current. Transfection with LTC subunits produced functional Ca(v)1.3 current from round cells, providing a homogeneous model system compared to native MSNs for studying D(2)R pathways. However, neither endogenous nor recombinant Ca(v)1.3 current was modulated by the D(2)R agonist quinpirole. We confirmed D(2)R expression in ST14A cells and also detected D(1)Rs, D(4)Rs, D(5)Rs, G(q), calcineurin and phospholipase A2 using RT-PCR and/or Western blot analysis. Phospholipase C β-1 (PLCβ-1) expression was not detected by Western blot analysis which may account for the lack of LTC modulation by D2Rs. These findings raise caution about the assumption that the presence of G-protein coupled receptors in cell lines indicates the presence of complete signaling cascades. However, exogenous arachidonic acid inhibited recombinant Ca(v)1.3 current indicating that channels expressed in ST14A cells are capable of modulation since they respond to a known signaling molecule downstream of D(2)Rs. Thus, ST14A cells provide a MSN-like cell line for studying channel modulation and signaling pathways that do not involve activation of PLCβ-1.

No MeSH data available.


Related in: MedlinePlus

Expression of D2R and M1R signaling pathway proteins in ST14A cells.(A) Top: D2R expression in ST14A cells, A9L cells (positive control) and tissues: SCG (superior cervical ganglia), STR (striatum, positive control), CTX (cortex). The predicted molecular weight (MW) for the D2R is 50 kDa. The 75 kDa form may represent a glycosylated form of the D2R versus long and short D2R splice variants, which differ by ~40 amino acids and would not account for this MW difference. Middle: M1R expression in ST14A or A9L cells. The predicted MW of M1Rs is 50 kDa. Bottom: Gqα expression in both cell lines and brain tissues. The predicted MW for Gqα is 42 kDa. (B) PLCβ-1 expression, or absence, in SCG, A9L or ST14A cells at various temperatures. The antibody recognizes PLCβ-1 fragments at 100 and 41 kDa. (M, lane for MW ladder) (C) The presence of cPLA2 in SCG neurons at postnatal day 1 (P1) or 4 (P4) or adult (A), A9L and ST14A cells under varying conditions. The predicted MW for cPLA2 is 110 kDa. β-actin, MW of 42 kDa, is shown as a loading control. (D) PP2B in ST14A cells loaded at varying concentrations; predicted MW of the α-subunit is 61 kDa.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492559&req=5

pone.0132469.g005: Expression of D2R and M1R signaling pathway proteins in ST14A cells.(A) Top: D2R expression in ST14A cells, A9L cells (positive control) and tissues: SCG (superior cervical ganglia), STR (striatum, positive control), CTX (cortex). The predicted molecular weight (MW) for the D2R is 50 kDa. The 75 kDa form may represent a glycosylated form of the D2R versus long and short D2R splice variants, which differ by ~40 amino acids and would not account for this MW difference. Middle: M1R expression in ST14A or A9L cells. The predicted MW of M1Rs is 50 kDa. Bottom: Gqα expression in both cell lines and brain tissues. The predicted MW for Gqα is 42 kDa. (B) PLCβ-1 expression, or absence, in SCG, A9L or ST14A cells at various temperatures. The antibody recognizes PLCβ-1 fragments at 100 and 41 kDa. (M, lane for MW ladder) (C) The presence of cPLA2 in SCG neurons at postnatal day 1 (P1) or 4 (P4) or adult (A), A9L and ST14A cells under varying conditions. The predicted MW for cPLA2 is 110 kDa. β-actin, MW of 42 kDa, is shown as a loading control. (D) PP2B in ST14A cells loaded at varying concentrations; predicted MW of the α-subunit is 61 kDa.

Mentions: After confirming ST14A cells exhibit a similar mRNA expression profile for dopamine receptors and GAD as MSNs, we examined whether D2Rs and downstream signaling molecules were expressed in ST14A cells. Using Western blot analysis, D2R and M1R expression was confirmed in ST14A cells. D2R antibodies recognized a band at 50 kDa, the expected molecular weight of D2Rs (Fig 5A, top panel) for striatum and cortex (positive controls). A second band at 75 kDa, absent in the A9L cell line, most likely represents a glycosylated form of the receptor [41], further supporting the idea that the ST14A cells are similar to neurons in these brain regions regarding post-translational modification of proteins. Fig 5A (middle panel) shows that ST14A cells, but not A9L cells, express M1Rs, displayed as a 50 kDa band on Western blots. This antibody recognized M1R expression in SCG, striatum, and cortex [34]. Expression of Gqα (Fig 5A, bottom panel), which couples to M1Rs, was also detected in the cell lines and tissue samples at the predicted molecular weight of 42 kDa. These results show that ST14A cells express D2R, M1R and Gqα proteins.


Characterization of ST14A Cells for Studying Modulation of Voltage-Gated Calcium Channels.

Roberts-Crowley ML, Rittenhouse AR - PLoS ONE (2015)

Expression of D2R and M1R signaling pathway proteins in ST14A cells.(A) Top: D2R expression in ST14A cells, A9L cells (positive control) and tissues: SCG (superior cervical ganglia), STR (striatum, positive control), CTX (cortex). The predicted molecular weight (MW) for the D2R is 50 kDa. The 75 kDa form may represent a glycosylated form of the D2R versus long and short D2R splice variants, which differ by ~40 amino acids and would not account for this MW difference. Middle: M1R expression in ST14A or A9L cells. The predicted MW of M1Rs is 50 kDa. Bottom: Gqα expression in both cell lines and brain tissues. The predicted MW for Gqα is 42 kDa. (B) PLCβ-1 expression, or absence, in SCG, A9L or ST14A cells at various temperatures. The antibody recognizes PLCβ-1 fragments at 100 and 41 kDa. (M, lane for MW ladder) (C) The presence of cPLA2 in SCG neurons at postnatal day 1 (P1) or 4 (P4) or adult (A), A9L and ST14A cells under varying conditions. The predicted MW for cPLA2 is 110 kDa. β-actin, MW of 42 kDa, is shown as a loading control. (D) PP2B in ST14A cells loaded at varying concentrations; predicted MW of the α-subunit is 61 kDa.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492559&req=5

pone.0132469.g005: Expression of D2R and M1R signaling pathway proteins in ST14A cells.(A) Top: D2R expression in ST14A cells, A9L cells (positive control) and tissues: SCG (superior cervical ganglia), STR (striatum, positive control), CTX (cortex). The predicted molecular weight (MW) for the D2R is 50 kDa. The 75 kDa form may represent a glycosylated form of the D2R versus long and short D2R splice variants, which differ by ~40 amino acids and would not account for this MW difference. Middle: M1R expression in ST14A or A9L cells. The predicted MW of M1Rs is 50 kDa. Bottom: Gqα expression in both cell lines and brain tissues. The predicted MW for Gqα is 42 kDa. (B) PLCβ-1 expression, or absence, in SCG, A9L or ST14A cells at various temperatures. The antibody recognizes PLCβ-1 fragments at 100 and 41 kDa. (M, lane for MW ladder) (C) The presence of cPLA2 in SCG neurons at postnatal day 1 (P1) or 4 (P4) or adult (A), A9L and ST14A cells under varying conditions. The predicted MW for cPLA2 is 110 kDa. β-actin, MW of 42 kDa, is shown as a loading control. (D) PP2B in ST14A cells loaded at varying concentrations; predicted MW of the α-subunit is 61 kDa.
Mentions: After confirming ST14A cells exhibit a similar mRNA expression profile for dopamine receptors and GAD as MSNs, we examined whether D2Rs and downstream signaling molecules were expressed in ST14A cells. Using Western blot analysis, D2R and M1R expression was confirmed in ST14A cells. D2R antibodies recognized a band at 50 kDa, the expected molecular weight of D2Rs (Fig 5A, top panel) for striatum and cortex (positive controls). A second band at 75 kDa, absent in the A9L cell line, most likely represents a glycosylated form of the receptor [41], further supporting the idea that the ST14A cells are similar to neurons in these brain regions regarding post-translational modification of proteins. Fig 5A (middle panel) shows that ST14A cells, but not A9L cells, express M1Rs, displayed as a 50 kDa band on Western blots. This antibody recognized M1R expression in SCG, striatum, and cortex [34]. Expression of Gqα (Fig 5A, bottom panel), which couples to M1Rs, was also detected in the cell lines and tissue samples at the predicted molecular weight of 42 kDa. These results show that ST14A cells express D2R, M1R and Gqα proteins.

Bottom Line: Transfection with LTC subunits produced functional Ca(v)1.3 current from round cells, providing a homogeneous model system compared to native MSNs for studying D(2)R pathways.However, neither endogenous nor recombinant Ca(v)1.3 current was modulated by the D(2)R agonist quinpirole.Thus, ST14A cells provide a MSN-like cell line for studying channel modulation and signaling pathways that do not involve activation of PLCβ-1.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Program in Neuroscience, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

ABSTRACT
In medium spiny neurons (MSNs) of the striatum, dopamine D2 receptors (D2Rs) specifically inhibit the Ca(v)1.3 subtype of L-type Ca(2+) channels (LTCs). MSNs are heterogeneous in their expression of dopamine receptors making the study of D2R pathways difficult in primary neurons. Here, we employed the ST14A cell line, derived from embryonic striatum and characterized to have properties of MSNs, to study Ca(v)1.3 current and its modulation by neurotransmitters. Round, undifferentiated ST14A cells exhibited little to no endogenous Ca(2+) current while differentiated ST14A cells expressed endogenous Ca(2+) current. Transfection with LTC subunits produced functional Ca(v)1.3 current from round cells, providing a homogeneous model system compared to native MSNs for studying D(2)R pathways. However, neither endogenous nor recombinant Ca(v)1.3 current was modulated by the D(2)R agonist quinpirole. We confirmed D(2)R expression in ST14A cells and also detected D(1)Rs, D(4)Rs, D(5)Rs, G(q), calcineurin and phospholipase A2 using RT-PCR and/or Western blot analysis. Phospholipase C β-1 (PLCβ-1) expression was not detected by Western blot analysis which may account for the lack of LTC modulation by D2Rs. These findings raise caution about the assumption that the presence of G-protein coupled receptors in cell lines indicates the presence of complete signaling cascades. However, exogenous arachidonic acid inhibited recombinant Ca(v)1.3 current indicating that channels expressed in ST14A cells are capable of modulation since they respond to a known signaling molecule downstream of D(2)Rs. Thus, ST14A cells provide a MSN-like cell line for studying channel modulation and signaling pathways that do not involve activation of PLCβ-1.

No MeSH data available.


Related in: MedlinePlus