tacrolimus and 2-2--3-5--6-pentachlorobiphenyl

ortho-Substituted PCBs mobilize Ca2+ from isolated brain microsomes by interaction with FKBP12/RyR complexes. Investigation into the cellular importance of this mechanism was undertaken using PC12 cells by fluoroimaging the actions of specific PCB congeners on [Ca2+]i and pH. RyR and IP3R share a common intracellular Ca2+ store in PC12 cells. Perfusion of nM to low microM PCB95 caused a transient rise of [Ca2+]i that was not completely dependent on extracellular Ca2+. Pre-incubation of the cells with ryanodine or FK506 completely eliminated PCB95 responses, suggesting a primary action on the FKPP12/RyR-sensitive store. PCB95, but not PCB126, induced a gradual decrease in cytosolic pH that could be completely eliminated by FK506 pre-incubation of the cells. Direct respiration measurement using isolated brain mitochondria demonstrated that neither of the PCBs directly altered any stage of mitochondrial respiration. These results revealed that PCB95 disrupts intracellular Ca2+ signaling in PC12 cells by interaction with the FKBP12/RyR complex that in turn accelerated cellular metabolism, possibly affecting signaling between ER and mitochondria. Since ortho-substituted PCBs have been shown to be neurotoxic and may affect neurodevelopment, studies on the molecular mechanism by which they alter cellular signaling may provide valuable information on the physiological roles of FKPB12 and RyR on neuronal functions.

Topics: Acidosis; Animals; Bradykinin; Brain Chemistry; Calcium; Calcium Signaling; Cell Respiration; Enzyme Inhibitors; Estrogen Antagonists; Hydrogen-Ion Concentration; Immunophilins; Inositol Phosphates; Intracellular Fluid; Male; Mitochondria; PC12 Cells; Pheochromocytoma; Polychlorinated Biphenyls; Rats; Rats, Sprague-Dawley; Ryanodine; Structure-Activity Relationship; Tacrolimus

Ortho-substituted polychlorinated biphenyls (PCBs) have been shown to alter microsomal Ca2+ transport by selective interaction with ryanodine receptors (RyRs) of muscle sarcoplasmic reticulum (SR) and brain endoplasmic reticulum. The mechanism underlying the actions of PCBs on Ca2+ transport is further elucidated with skeletal SR enriched in Ry1R. Disruption of the association between immunophilin FKBP12 and Ry1R with FK 506 or rapamycin completely eliminates PCB 95-enhanced binding of [3H]ryanodine (IC50 approximately 35 microM) to Ry1R and PCB 95-induced release of Ca2+ from actively loaded SR vesicles (IC50 approximately 11 microM), demonstrating a FKBP12-dependent mechanism. FK 506 selectively eliminates PCB 95-induced Ca2+ release from SR because Ry1R maintains responsiveness to caffeine and Ca2+. PCB 95 and FK 506 are used to examine the relationship between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive Ca2+ leak pathways present in SR vesicles. Micromolar ryanodine completely blocks ryanodine-sensitive Ca2+ efflux but neither eliminates the ryanodine-insensitive Ca2+ leak unmasked by thapsigargin nor enhances the loading capacity of SR vesicles. PCB 95 alone enhances thapsigargin evoked Ca2+ release and therefore diminishes the loading capacity of SR vesicles. However, in the presence of micromolar ryanodine, PCB 95 dose-dependently eliminates the Ca2+ leak unmasked by thapsigargin and significantly enhances the loading capacity of SR vesicles. The actions of PCB 95 on SR-loading capacity are additive with those of FK 506. Structural specificity for these novel actions are further demonstrated with coplanar PCB 126, which is inactive toward Ry1R and lacks the ability to alter the Ca2+ leak pathway. The results reveal that FKBP12 relates ryanodine-insensitive Ca2+ "leak" and ryanodine-sensitive Ca2+ channel efflux pathways of SR by modulating distinct conformations Ry1R complexes. Noncoplanar PCBs, like PCB 95, alter SR Ca2+ buffering by an FKBP12-mediated mechanism. An immunophilin-based mechanism could account for the toxic actions attributed to certain noncoplanar PCB congeners.

Topics: Animals; Caffeine; Calcium; Calcium Channels; Carrier Proteins; DNA-Binding Proteins; Drug Interactions; Heat-Shock Proteins; Ion Transport; Microsomes; Polychlorinated Biphenyls; Polyenes; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Sirolimus; Tacrolimus; Tacrolimus Binding Proteins