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Subtypes of alpha 1-adrenoceptors in DDT1 MF-2 and BC3H-1 clonal cell lines
C Han1, T A Esbenshade, K P Minneman
1Department of Pharmacology, Emory University Medical School, Atlanta, GA 30322.
Abstract:
We examined which subtype(s) of alpha 1-adrenoceptors are expressed in the widely used DDT1 MF-2 and BC3H-1 cell lines. Pretreatment with chloroethylclonidine (CEC) inactivated 76-85% of the specific [125I]BE 2254 binding sites in membrane preparations from both cell lines. Competition with subtype-selective competitive antagonists showed primarily the alpha 1B subtype in both cell lines. However, in BC3H-1 cells 5-methyl-urapidil showed complex behavior suggesting that about half of the binding sites had a lower affinity. Chloroethylclonidine pretreatment eliminated [3H]inositol phosphate responses to norepinephrine in both cell lines. Measurement of intracellular Ca2+ with fura-2 in DDT1 MF-2 cells showed that norepinephrine induced a complex response involving both transient and sustained components. Chloroethylclonidine pretreatment blocked both responses, while chelation of extracellular Ca2+ left the transient response intact but eliminated the sustained component. These results support previous work that these cell lines contain alpha 1B-adrenoceptors linked to inositol phosphate formation and mobilization of intracellular Ca2+. However, these results show that alpha 1B-adrenoceptors can be linked to Ca2+ influx as well as intracellular mobilization, and support the existence of pharmacologically distinct alpha 1B variants.
Insights
This study identifies alpha 1B-adrenoceptors in DDT1 MF-2 and BC3H-1 cells, revealing their role in calcium signaling and potential for distinct functional variants.
Area of Science:
- Pharmacology
- Cell Biology
- Adrenoceptor Research
Background:
- DDT1 MF-2 and BC3H-1 cell lines are widely used models.
- Understanding alpha 1-adrenoceptor subtypes is crucial for drug development.
Purpose of the Study:
- To determine the specific alpha 1-adrenoceptor subtypes expressed in DDT1 MF-2 and BC3H-1 cells.
- To investigate the signaling pathways linked to these receptors, particularly calcium mobilization.
Main Methods:
- Radioligand binding assays using [125I]BE 2254.
- Competition assays with subtype-selective antagonists.
- Measurement of inositol phosphate formation and intracellular calcium levels (using fura-2).
- Pharmacological manipulation with chloroethylclonidine (CEC) and extracellular calcium chelation.
Main Results:
- Both cell lines predominantly express the alpha 1B-adrenoceptor subtype.
- CEC treatment inactivated a significant portion of binding sites and blocked norepinephrine-induced responses.
- Norepinephrine triggered complex intracellular calcium responses, involving both transient and sustained phases.
- The sustained calcium response was dependent on extracellular calcium influx, while the transient response involved intracellular stores.
Conclusions:
- DDT1 MF-2 and BC3H-1 cells express alpha 1B-adrenoceptors linked to inositol phosphate formation and intracellular calcium release.
- Alpha 1B-adrenoceptors can also mediate calcium influx, suggesting the existence of pharmacologically distinct alpha 1B variants.
- These findings enhance our understanding of alpha 1B-adrenoceptor signaling diversity.