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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

ST14A cells express several D2R family mRNAs.ST14A mRNA transcripts were amplified by RT-PCR. (A) D2R mRNA expression in ST14A, A9L, HEK 293 cells and in striatum. A9L cells that express D2Rs and striatum served as positive controls. Left lane contains a 100 bp ladder; brightest band is 600 bp. The band for ST14A cells was sequenced and BLAST search results matched previously published sequences for D2Rs. No D2R bands were detected in HEK 293 cells. (B) GAD65/67 mRNA expression in ST14A, striatum, cortex, A9L and HEK 293 cells. As a control, ST14A mRNA was not reverse-transcribed (- RT).
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pone.0132469.g004: ST14A cells express several D2R family mRNAs.ST14A mRNA transcripts were amplified by RT-PCR. (A) D2R mRNA expression in ST14A, A9L, HEK 293 cells and in striatum. A9L cells that express D2Rs and striatum served as positive controls. Left lane contains a 100 bp ladder; brightest band is 600 bp. The band for ST14A cells was sequenced and BLAST search results matched previously published sequences for D2Rs. No D2R bands were detected in HEK 293 cells. (B) GAD65/67 mRNA expression in ST14A, striatum, cortex, A9L and HEK 293 cells. As a control, ST14A mRNA was not reverse-transcribed (- RT).

Mentions: A non-selective D2R-like agonist, Quin also activates D3 and D4Rs [38]. Subsequently, D3 and D4Rs modulate immediate early gene expression [39, 40] raising the question of the identity of dopamine receptors in ST14A cells. Using RT-PCR, we examined dopamine receptor (D1, D2, D3, D4, and D5) mRNA content in ST14A cells, striatum (positive control for all dopamine receptors), cortex, A9L cells (D2R positive control; see Materials and Methods) or HEK 293 cells (negative control for all dopamine receptors). PCR products were detected for long and short splice variants of the D2R in ST14A cells (n = 4/11 and 2/11, respectively, striatum (n = 1/2 and 2/2, respectively), cortex (n = 1/2 for both) and A9L cells (n = 2/5 and 2/5, respectively) as shown in Fig 4A. Additionally, D1R (n = 2/10), D4R (7/10), D5R (4/10) (data not shown) mRNAs were also detected in ST14A cells, regardless of the temperature at which cells were grown. D1R, D4R and D5R mRNAs were detected in striatum whereas D1R and D4R mRNAs were detected in the cortex (data not shown). D4R mRNA was also detected in A9L cells (n = 3/4). Experiments for D3R mRNA expression in striatum, cortex, and ST14A cell samples resulted in a smear despite several attempts to adjust the protocol. HEK 293 cells showed no expression of any of the dopamine receptors tested. These results show that ST14A cells express mRNA for more than one D2-like receptor; this finding could account for the previously reported D2R-like changes in pCREB [29].


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

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

ST14A cells express several D2R family mRNAs.ST14A mRNA transcripts were amplified by RT-PCR. (A) D2R mRNA expression in ST14A, A9L, HEK 293 cells and in striatum. A9L cells that express D2Rs and striatum served as positive controls. Left lane contains a 100 bp ladder; brightest band is 600 bp. The band for ST14A cells was sequenced and BLAST search results matched previously published sequences for D2Rs. No D2R bands were detected in HEK 293 cells. (B) GAD65/67 mRNA expression in ST14A, striatum, cortex, A9L and HEK 293 cells. As a control, ST14A mRNA was not reverse-transcribed (- RT).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132469.g004: ST14A cells express several D2R family mRNAs.ST14A mRNA transcripts were amplified by RT-PCR. (A) D2R mRNA expression in ST14A, A9L, HEK 293 cells and in striatum. A9L cells that express D2Rs and striatum served as positive controls. Left lane contains a 100 bp ladder; brightest band is 600 bp. The band for ST14A cells was sequenced and BLAST search results matched previously published sequences for D2Rs. No D2R bands were detected in HEK 293 cells. (B) GAD65/67 mRNA expression in ST14A, striatum, cortex, A9L and HEK 293 cells. As a control, ST14A mRNA was not reverse-transcribed (- RT).
Mentions: A non-selective D2R-like agonist, Quin also activates D3 and D4Rs [38]. Subsequently, D3 and D4Rs modulate immediate early gene expression [39, 40] raising the question of the identity of dopamine receptors in ST14A cells. Using RT-PCR, we examined dopamine receptor (D1, D2, D3, D4, and D5) mRNA content in ST14A cells, striatum (positive control for all dopamine receptors), cortex, A9L cells (D2R positive control; see Materials and Methods) or HEK 293 cells (negative control for all dopamine receptors). PCR products were detected for long and short splice variants of the D2R in ST14A cells (n = 4/11 and 2/11, respectively, striatum (n = 1/2 and 2/2, respectively), cortex (n = 1/2 for both) and A9L cells (n = 2/5 and 2/5, respectively) as shown in Fig 4A. Additionally, D1R (n = 2/10), D4R (7/10), D5R (4/10) (data not shown) mRNAs were also detected in ST14A cells, regardless of the temperature at which cells were grown. D1R, D4R and D5R mRNAs were detected in striatum whereas D1R and D4R mRNAs were detected in the cortex (data not shown). D4R mRNA was also detected in A9L cells (n = 3/4). Experiments for D3R mRNA expression in striatum, cortex, and ST14A cell samples resulted in a smear despite several attempts to adjust the protocol. HEK 293 cells showed no expression of any of the dopamine receptors tested. These results show that ST14A cells express mRNA for more than one D2-like receptor; this finding could account for the previously reported D2R-like changes in pCREB [29].

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