Effects of simple aromatic compounds and flavonoids on Ca2+ fluxes in rat pituitary GH4C1 cells
Introduction
Only plants and microorganisms are capable of biologically synthesizing the aromatic nucleus, which is a base structure in plant phenolic compounds produced via the shikimic acid pathway. For example, flavonoids are polyphenolic substances that are based on a flavan nucleus consisting of 15 carbon atoms arranged in three rings (C6–C3–C6, see Fig. 1). The effects of flavonols and flavones on the enzymes involved in the regulation of cell division and proliferation, platelet aggregation, detoxification, as well as inflammatory and immune responses have been described (Middleton and Kandaswami, 1994). Phenolic compounds have been reported to interfere with various stages of cancer development Stavric, 1995, Huang and Ferraro, 1992, and a number of naturally occurring as well as synthetic flavonoids have been shown to have potent anti-human immunodeficiency virus (HIV) activity in vitro (Wang et al., 1998). Because of their phytoestrogenic properties, flavonoids, as well as other plant phenolic compounds, have also been implicated in preventing menopausal symptoms, osteoporosis, breast and ovarian cancer, as well as heart disease Adlerkreutz et al., 1992, Kurzer and Xu, 1997. In vivo studies suggest that especially flavonoids may reduce the risk of coronary disease Fotsis et al., 1993, Kapiotis et al., 1997. In fact, epidemiological studies indicate a beneficial role of flavonoids in diminishing the risk of coronary heart disease Hertog et al., 1993, Hollman et al., 1996.
The effects of flavonoids on the cardiovascular system has been attributed to a modulation of Ca2+ homeostasis. The antagonistic activity of flavonoids and other plant phenolic compounds has usually been investigated by measuring the inhibition of depolarization-induced contractions of smooth muscle preparations (Vuorela et al., 1997). These contractions are apparently evoked by Ca2+-dependent mechanisms and are indeed inhibited by Ca2+ channel antagonists (Hof and Vuorela, 1983). The L-type voltage-operating Ca2+ channels (VOCCs, i.e. slowly inactivating VOCCs) are of crucial importance in the regulation of excitation–contraction coupling in cardiac and vascular smooth muscle (Tsien and Tsien, 1990). Several flavonoids modulate protein tyrosine kinase, protein kinase A and C dependent pathways in different cell systems Ferriola et al., 1989, Duarte et al., 1993a, Agullo et al., 1997, Revuelta et al., 1997. Inhibition of protein tyrosine kinase by genistein has been shown to inhibit voltage-gated potassium channels (Smirnov and Aaronson, 1995), and retinal cyclic nucleotide-gated channels are inhibited as a result of inhibition of protein tyrosine kinase (Mergler et al., 1998). In pituitary cells, several intracellular pathways (e.g., protein kinase A, protein kinase C and protein tyrosine kinase) modulate the activity of the VOCCs, and thus a multitude of Ca2+-dependent physiological processes, e.g. hormone secretion and synthesis Tan and Tashjian, 1984, Albert and Tashjian, 1984, Cataldi et al., 1996.
The antagonistic activity of certain plant phenolic compounds has long been acknowledged. The previous works have usually been focused on depolarization-induced contraction in smooth muscle preparations. The effects have been reported to be related to Ca2+ fluxes or metabolism. VOCCs can be seen as a target of intracellular pathways, e.g. protein kinase A, protein kinase C and protein tyrosine kinase. The effects of plant phenolic compounds suggest that these compounds may regulate Ca2+ fluxes in several endocrine glands. Therefore, the aim of the present study was to screen the effects of simple aromatic compounds and different classes of flavonoids on high K+-evoked entry of 45Ca2+ in well-characterised clonal rat GH4C1 pituitary cells.
Section snippets
Materials
The culture medium, serum, lanthanium chloride, and penicillin–streptomycin used for cell culture were purchased from Gibco BRL (UK) and Sigma (MO, USA). Dulbecco's phosphate-buffered saline (PBS) was from Gibco BRL, and EDTA from Sigma. Falcon tissue culture dishes of Ø 35 mm (3001) and Ø 100 mm (3003) were obtained from Becton Dickinson (UK). All other chemicals were of reagent grade. 45CaCl2 (2.0 mCi/ml) was purchased from Pharmacia Amersham Biotech (UK), and Optiphase Hisafe 2 liquid
Inhibitory effect on 45Ca2+ uptake
Recent studies have suggested that isoflavonoids may inhibit VOCCs Wijetunge et al., 1992, Wijetunge and Hughes, 1995. We found that several phenolic compounds had a clear Ca2+ antagonistic activity similar to that of the therapeutically used Ca2+ channel antagonist verapamil hydrochloride. The flavone aglycones luteolin and flavone produced a 51.4% and a 63.5% inhibition at the highest tested concentration (20 μg/ml). Flavones with sugar moieties had the weakest inhibitory effect on 45Ca2+
Discussion
This is the first time simple aromatic compounds and flavonoids from different subgroups have been systematically screened in order to compare the relative potencies of the compounds on VOCCs, using cultivated rat pituitary GH4C1 cells as a model. The rationale for using GH4C1 cells is that the calcium channels in these cells have been thoroughly characterized and the cells have been used in a multitude of studies on calcium channels (e.g., Xi et al., 1992, Fu et al., 1997, Peri et al., 2000),
Acknowledgments
This study was supported by grants from the Academy of Finland, the National Technology Agency in Finland, the Emil Aadltonen Foundation, and the Research Foundation of Farmos in Finland.
References (44)
- et al.
Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition
Biochem. Pharmacol.
(1997) - et al.
Genistein, a specific inhibitor of tyrosine-specific protein kinases
J. Biol. Chem.
(1987) - et al.
Relationship of thyrotropin-releasing hormone-induced spike and plateau phases in cytosolic free Ca2+ concentrations to hormone secretion. Selective blockade using ionomycin and nifedipine
J. Biol. Chem.
(1984) - et al.
Protein-tyrosine kinases activate while protein-tyrosine phosphatases inhibit L-type calcium channel activity in pituitary GH3 cells
J. Biol. Chem.
(1996) - et al.
Vasodilatory effects of flavonoids in rat aortic smooth muscle: structure–activity relationships
Gen. Pharmacol.
(1993) - et al.
Vasodilator effects of quercetin in isolated rat vascular smooth muscle
Eur. J. Pharmacol.
(1993) - et al.
Protein kinase C inhibition by flavonoids. Kinetic mechanisms and structure activity relationships
Biochem. Pharmacol.
(1989) - et al.
R24571: a new powerful inhibitor of red blood cell Ca2+-transport ATPase and of calmodulin-regulated function
Biochem. Biophys. Res. Commun.
(1981) - et al.
Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen elderly study
Lancet
(1993) - et al.
Assessing calcium antagonism on vascular smooth muscle: a comparison of three methods
J. Pharmacol. Methods
(1983)
Cyclic nucleotide phosphodiesterase inhibitors prevent aggregation of human platelets by raising cyclic AMP and reducing cytoplasmic free calcium mobilization
Thromb. Res.
Depolarization-dependent effect of flavonoids in rat uterine smooth muscle contraction elicited by CaCl2
Gen. Pharmacol.
Voltage-dependent calcium channels in pituitary cells in culture: I. Characterization by 45Ca2+ fluxes
J. Biol. Chem.
Clonal strains of hormone-producing pituitary cells
Methods Enzymol.
Sphingosine inhibits voltage-operated calcium channels in GH4C1 cells
J. Biol. Chem.
pp60c-src increase voltage-operated calcium channel currents in vascular smooth muscle cells
Biochem. Biophys. Res. Commun.
Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells
Biochem. Biophys. Res. Commun.
Maitotoxin induces a calcium-dependent membrane depolarization in GH4C1 pituitary cells via activation of type L voltage-dependent calcium channels
J. Biol. Chem.
Effects of 3,3′,-di-O-methylquercetin on guinea-pig isolated smooth muscle
J. Pharm. Pharmacol.
Dietary phytoestrogens and cancer: in vitro and in vivo studies
J. Steroid Biochem. Mol. Biol.
Non-selective inhibition of mammalian protein kinases by flavonoids in vitro
Res. Commun. Chem. Pathol. Pharmacol.
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