Short communicationEvaluation of β-adrenergic receptor subtypes in the human prostate cancer cell line-LNCaP
Introduction
Prostate tissue has been demonstrated to be a rich source of α1- and β-AR [1]. Biochemical studies have shown that β-AR agonists are able to stimulate adenylyl cyclase and raise cAMP levels in the rat prostate [2], [3]. Accumulation of cAMP and differentiation of neuroendocrine cells have been observed in human prostate cancer cell lines treated with forskolin, dibutyryl cAMP, and catecholamine-like agonists (isoproterenol and epinephrine) [4]. These effects were reversible upon withdrawal of these agents [5]. The pharmacological properties of β-AR agonists with respect to receptor binding and cAMP generation were examined in PC-3 cells, a human prostate androgen-independent cell line [6]. The results of these studies indicated that PC-3 cells contained a large population of β2-ARs. The role of β-AR subtypes involved in catecholamine-mediated cAMP elevations in a human lymph node carcinoma of the prostate (LNCaP cells), an androgen-sensitive cell line, has not been elucidated.
In this study, we used a reporter gene assay to investigate the effects of catecholamines and known β-AR subtype selective agonists on cAMP levels in LNCaP cells. The pharmacological specificity of agonist actions in this cell line was characterized by the use of non-selective and selective β-AR antagonists.
Section snippets
Cell culture
LNCaP cells were purchased from the American Type Culture Collection. These cells were grown in 150×25 mm petri dishes with a 1:1 mixture of Dulbecco’s modified Eagle’s medium/Ham’s F12 medium (DMEM/F12) supplemented with 10% fetal bovine serum, penicillin G sodium (100 U/mL), or streptomycin (100 μg/mL). When the cultures were 75% confluent, the cells were detached by trypsinization (0.05% trypsin EDTA for 1 min). Cell culture reagents were obtained from Life Technologies. The compounds used in
Results and discussion
Initial experiments in LNCaP cells showed that forskolin (20 μM) produced on the average a 92-fold induction of luciferase gene expression. This indicates that adenylyl cyclase is involved in the transcriptional activation of the 6 CRE-LUC construct. Subsequent experiments with catecholamines generated concentration–response curves for isoproterenol, epinephrine, and norepinephrine (Fig. 1A) with ec50 values of 8.31, 16.2, and 77.5 nM, respectively. The corresponding pec50 values (mean±SEM) for
Acknowledgements
This work was supported, in part, by the USDA, ARS Specific Cooperative Agreement No. #58-6408-2-0009. R.D. Salas thanks the Department of Pharmacology, Universidad Central de Venezuela (Caracas, Venezuela), and the Department of Pharmacology, Universidad de Cartagena (Cartagena, Colombia), for financial assistance.
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