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Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP₃ receptor function.

ABSTRACT: Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca²⁺) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Ca_v blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca²⁺]_cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca²⁺ pools, and Ca²⁺ influx by ORAI channels have been associated with the increase in [Ca²⁺]_cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil's effect on [Ca²⁺]_cyt. To address these questions, we monitored changes in [Ca²⁺]_cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca²⁺]_cyt at concentrations above 10 μM (IC₅₀ around 50 μM) and a fast Ca²⁺ increase capacity at 100 μM. We found that inhibiting IP₃ receptors, depleting the ER-Ca²⁺ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca²⁺]_cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca²⁺ influx by store-operated Ca²⁺ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP₃R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

SUBMITTER: Souza Bomfim GH

PROVIDER: S-EPMC8607413 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Intracellular Ca2+ is critical for regulating airway smooth muscle (ASM) tension. A rapid rise in the intracellular Ca2+ concentration ([Ca2+]i) of ASM cells is crucial for modulating the intensity and length of the ASM contraction. Because this rapid increase in [Ca2+]i largely depends on the balance between Ca2+ released from intracellular Ca2+ stores and extracellular Ca2+ entry, exploring the mechanisms mediating Ca2+ transport is critical for understanding ASM contractility and the pathogenesis of bronchial contraction disorders. Transient receptor potential vanilloid 4 (TRPV4) is a highly Ca2+-permeable non-selective cation channel that mediates Ca2+ influx to increase [Ca2+]i, which then directly or indirectly regulates the contraction and relaxation of ASM. The [Ca2+]i returns to basal levels through several uptake and extrusion pumps, such as the sarco(endo)plasmic reticulum Ca2+ ATPase and inositol 1,4,5-trisphosphate receptors (IP3Rs), the plasmalemmal Ca2+ ATPase, and the plasma membrane Na+/Ca2+ exchanger (NCX). Thus, to further understand ASM tension regulation in normal and diseased tissue, the present study examined whether an interaction exists among TRPV4, IP3Rs, and NCX. The TRPV4-specific and potent agonist GSK1016790A increased [Ca2+]i in mouse ASM cells, an effect that was completely blocked by the TRPV4-specific antagonist HC067047. However, GSK1016790A induced relaxation in mouse tracheal rings precontracted with carbachol in vitro. To determine the mechanism underlying this TRPV4-induced relaxation of ASM, we blocked specific downstream molecules. We found that the GSK1016790A-induced relaxation was abolished by the NCX inhibitors KB-R7943 and LiCl but not by specific inhibitors of the Ca2+-activated large-, intermediate-, or small-conductance K+ channels (BKCa, IK, and SK3, respectively). The results of co-immunoprecipitation (co-IP) assays showed an interaction of TRPV4 and IP3R1 with NCXs. Taken together, these findings support a physical and functional interaction of TRPV4 and IP3R1 with NCXs as a novel TRPV4-mediated Ca2+ signaling mechanism and suggest a potential target for regulation of ASM tension and treatment of respiratory diseases, especially tracheal spasm.

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Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP3 receptor function.

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Mibefradil alters intracellular calcium concentration by activation of phospholipase C and IP₃ receptor function.