Tauroursodeoxycholate (TUDC) prevents the cholestatic effects of estradiol 17ß-D-glucuronide (E17G) via activation of Ca2+/calmodulin protein kinase II (CaMKII).

MEDEOT, ANABELA CAROLINA; BOAGLIO, ANDREA C.; RAZORI, MARÍA VALERIA; SALAS, GIMENA; SCHUCK, VIRGINIA SOLEDAD; CROCENZI, FERNANDO ARIEL; ROMA, MARCELO GABRIEL

Reunión virtual

Congreso; Reunión de la Sociedad de Biociencias (SAIC-SAI-SAFIS) 2020; 2020

SAIC-SAI-SAFIS

E17G, the main etiologic agent of pregnancy-inducedcholestasis, triggers bile flow drop by inducing endocytosis and intracellularretention of the canalicular transporters involved in bile secretion Mrp2 andBsep, via activation of cPKC and PI3K, respectively. We showed that TUDC, theactive metabolite of ursodeoxycholic acid (the most effective drug used to treat this condition), counteractsall these pathomechanisms. However, the signaling mediators involved in thisprotection remains unknown. Since TUDC increases cytosolic Ca2+ and,by doing so, promotes vesicular exocytosis under cholestatic conditions, wetested in rat hepatocyte couplets (RHC) whether Ca2+ and itsdownstream effector CaMKII mediate TUDC protective effects. E17G (200 µM, 20min) decreased by 56±1 % and 60±2 % (p<0.05) the % of RHC accumulating apicallythe fluorescent Bsep and Mrp2 substrates cholyl-lysilfluorecein and GSH-S-methylflourescein,respectively, as assessed by inverted fluorescence microscopy. Pretreatmentwith TUDC (100 µM, 15 min) fully prevented these alterations. Beneficialeffects of TUDC were fully blocked by the Ca2+ sequestering agentBAPTA (20 µM) and by the CaMKII inhibitor KN62 (10 µM). E17G-induced endocytosisof Mrp2, assessed by immunostaining followed by confocal microscopy and imageanalysis, was significantly prevented by TUDCA, and this was fully halted byBAPTA and KN62. Western blot analysis revealed that TUDC impeded the translocationof the main cPKC isoform, PKC-a,to plasma membrane and prevented phosphorylation of Akt, two events indicativeof activation of these pro-cholestatic signaling molecules, and that these activationswere fully blocked by BAPTA and KN62. We conclude that TUDC-inducedpreservation of Bsep and Mrp2 function and location in E17G-induced cholestasisinvolves intracellular Ca2+ elevations and further CaMKII activation,and that CaMKII crosstalks with the pro-cholestatic signaling pathways cPKC andPI3K/Akt to impede their activation.