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Decrease in thromboxane A2 receptor expression by differentiation with dibutyryl cyclic AMP in 1321N1 human
S Honma1, N Nakahata, H Kobayashi
1Department of Pharmaceutical Molecular Biology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Abstract:
Thromboxane A2 (TXA2) receptor expression with its signaling was investigated in 1321N1 human astrocytoma cells differentiated with dibutyryl cyclic AMP (dbcAMP). The cells cultured in 0.5% fetal calf serum containing 0.5 mM dbcAMP for 3 days showed the star-shaped morphology, accompanied with the reduction of a TXA2 mimetic U46619-induced phosphoinositide hydrolysis and Ca2+ mobilization. Immunoblotting analysis revealed that human astrocytoma cells expressed phospholipase C (PLC)-beta1 and -beta3, but not PLC-beta2. The contents of PLC-beta1 and beta3 were not changed by the differentiation. The alpha subunit of Gq/ll bound to TXA2-receptor was reduced by the differentiation, determined by immunoblotting after immunoprecipitation with an anti-TXA2-receptor antibody. Scatchard analysis of the binding of [3H]SQ29548, a TXA2 receptor antagonist, to the membranes revealed that the maximum binding site was reduced by the differentiation. The expression of TXA2 receptor mRNA also was reduced by the differentiation, determined by reverse-transcribed-polymerase chain reaction. Although placental type of TXA2 receptor mRNA expression increased after the differentiation, endothelial type of TXA2 receptor mRNA expression slightly decreased. The results suggest that 1321N1 human astrocytoma cells differentiated with dbcAMP show impaired TXA2 receptor-mediated phosphoinositide hydrolysis and Ca2+ mobilization, due to the decrease in TXA2 receptor number.
Insights
Dibutyryl cyclic AMP (dbcAMP) differentiation of 1321N1 astrocytoma cells reduces Thromboxane A2 (TXA2) receptor expression and signaling. This leads to impaired phosphoinositide hydrolysis and calcium mobilization, indicating decreased TXA2 receptor numbers.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Thromboxane A2 (TXA2) plays a role in cellular signaling pathways.
- Astrocytoma cells are a type of brain tumor cell.
- Understanding receptor expression and signaling is crucial for cellular function.
Purpose of the Study:
- To investigate Thromboxane A2 (TXA2) receptor expression and signaling in differentiated 1321N1 human astrocytoma cells.
- To determine the effect of dibutyryl cyclic AMP (dbcAMP) differentiation on TXA2 receptor-mediated cellular responses.
Main Methods:
- 1321N1 human astrocytoma cells were differentiated using dibutyryl cyclic AMP (dbcAMP).
- Assays were performed to measure phosphoinositide hydrolysis and Ca2+ mobilization.
- Immunoblotting, immunoprecipitation, Scatchard analysis, and reverse-transcribed-polymerase chain reaction were used to assess receptor and protein expression.
Main Results:
- Differentiated cells exhibited reduced TXA2 mimetic-induced phosphoinositide hydrolysis and Ca2+ mobilization.
- The number of TXA2 receptors and the expression of TXA2 receptor mRNA were decreased following differentiation.
- While placental-type TXA2 receptor mRNA increased, endothelial-type decreased slightly.
- Phospholipase C (PLC)-beta1 and -beta3 expression remained unchanged, but Gq/11 alpha subunit levels decreased.
Conclusions:
- Dibutyryl cyclic AMP (dbcAMP) differentiation of 1321N1 astrocytoma cells leads to a significant decrease in Thromboxane A2 (TXA2) receptor number.
- This reduction in TXA2 receptor expression results in impaired TXA2 receptor-mediated phosphoinositide hydrolysis and Ca2+ mobilization.
- The findings highlight the impact of differentiation on specific receptor signaling pathways in astrocytoma cells.