Effects of simple aromatic compounds and flavonoids on Ca2+ fluxes in rat pituitary GH4C1 cells

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Abstract

The biological activity of phenolic compounds from plants is well documented in vitro, but little is known about the possible effect of simple aromatic compounds and flavonoids on voltage-operated Ca2+ channels (VOCCs). In pituitary cells, several intracellular pathways may regulate the activity of VOCCs. In this study, we investigated the effect of nine phenylpropanes and metanes, and 20 flavonoids on high K+-induced 45Ca2+ entry in clonal rat pituitary GH4C1 cells. At the highest dose tested (20 μg/ml), flavone (a flavone) inhibited 45Ca2+ entry by 63.5%, naringenin (a flavanone) by 56.3% and genistein (an isoflavone) by 54.6%. The phenylmetane derivative octyl gallate was the most potent compound tested, with an IC50 value of 15.0 μg/ml. The IC50 value for the reference compound verapamil hydrochloride was 3.0 μg/ml. In sharp contrast to the above, the flavonols quercetin and morin potentiated 45Ca2+ entry. At 20 μg/ml, quercetin increased 45Ca2+ entry by 54.1% and morin by 48.0%. Quercetin increased the cellular cAMP content in a concentration-dependent manner. H 89, an inhibitor of protein kinase A, inhibited the effect of quercetin on 45Ca2+ entry. The results thus suggest that the effect of quercetin is the result of a protein kinase A-mediated activation of VOCCs. Quercetin induced a rapid and marked increase in both the transient (143.1±4.2%) and delayed (198.8±10.0%) Ca2+ currents, measured by the whole cell patch clamp technique. The onset of the inhibitory effect of octyl gallate was slow, but resulted in an almost complete inhibition of both Ca2+ currents.

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.

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