Effect of plant flavonoids on the volume regulation of rat thymocytes under hypoosmotic stress.

Abstract

Cell volume regulation and volume-regulated anion channels are critical for cell survival in non-isosmotic conditions, and dysregulation of this system is detrimental. Although genes and proteins underlying this basic cellular machinery were recently identified, the pharmacology remains poorly explored. We examined effects of 16 flavonoids on the regulatory volume decrease (RVD) of thymocytes under hypoosmotic stress assessed by light transmittance and on the activity of volume-sensitive chloride channel by patch-clamp technique. Comparison of effects of flavonoids on RVD revealed a group of four active substances with lehmannin being the strongest inhibitor (IC50 = 8.8 μM). Structure-functional comparison suggested that hydrophobicity brought about by methoxy, prenyl or lavandulyl groups as well as by the absence of glucosyl fragment together with localization of the phenyl ring B at the position C2 (which is at C3 in totally inactive isoflavones) are important structural determinants for the flavonoids activity as volume regulation inhibitors. All active flavonoids suppressed RVD under Gramicidin D-NMDG hypotonic stress conditions when cationic permeability was increased by an ionophore, gramicidin D, with all extracellular monovalent cations replaced with bulky NMDG+ suggesting that they target volume-sensitive anionic permeability. While effects of hispidulin and pulicarin were only partial, lehmannin and pinocembrin completely abolished RVD under Gramicidin D-NMDG conditions. In direct patch-clamp experiments, lehmannin and pinocembrin produced a strong inhibiting effect on the swelling-induced whole-cell chloride conductance in a voltage-independent manner. Lehmannin, pinocembrin, and possibly hispidulin and pulicarin may serve as leads for developing effective low-toxic immunomodulators.