Biphenols block calcium entry in response to activation of the M3 muscarinic receptor

Abstract

Cell‐permeable biphenols, such as honokiol, present in herbal teas, inhibit autonomic responses. The effects of biphenols on calcium signaling activated by human M3 muscarinic receptors was studied in Chinese hamster ovary (CHO) cells. Receptor binding was determined by radiolabelled ligand binding; intracellular calcium concentrations were determined using a fura‐2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay. Activation of M3 muscarinic receptors with carbachol induced a biphasic increase in [Ca2+]i: an initial, IP3‐ mediated release of Ca2+ from endoplasmic reticulum stores followed by a sustained phase of steady Ca2+ entry (i.e., store operated calcium entry, SOCE). Biphneols had potent (EC50 ≈ 5 μM) inhibitory effects on SOCE that was induced by the activation of the M3 receptor. SOCE was also examined by acute exposure to thapsigarin (2 μM) in a calcium‐free medium, followed by reintroduction of calcium. Again, SOCE was severely depressed by biphenols. The effect of honokiol on SOCE was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor‐mediated calcium release, or depletion of ER calcium stores. Moreover, biphenols did not disrupt the ligand binding properties of M3 receptors. An inhibition of SOCE probably contributes to biphenol disruption of parasympathetic functions.