sq-23377 and 2-tert-butylhydroquinone

Tert-butylhydroquinone (tBHQ) is a commonly used food preservative with known immunomodulatory activity; however, there is little information regarding its role on natural killer (NK) cell activation and function. tBHQ is a known activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which results in induction of cytoprotective genes. Activation of Nrf2 has been shown to modulate immune responses in a number of different models. In addition, studies in our laboratory have shown that tBHQ inhibits numerous early events following T cell activation. In the current study, we investigated whether activated NK cells are impacted by tBHQ, since many signaling cascades that control NK cell effector function also contribute to T cell function. Splenocytes were isolated from female, wild-type C57Bl/6J mice and treated with 1 μM or 5 μM tBHQ. NK cell function was assessed after activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 24 h. Activation of NK cells in the presence of tBHQ decreased total NK cell percentage, production of intracellular interferon gamma (IFNɣ), granzyme B, and perforin, and induction of the cell surface proteins CD25 and CD69, which are markers of NK cell activation. In addition to NK cell effector function, NK cell maturation was also altered in response to tBHQ. Notably, this is the first study to demonstrate that the Nrf2 activator, tBHQ, negatively impacts effector function and maturation of NK cells.

Topics: Animals; Antioxidants; Cells, Cultured; Female; Gene Expression Regulation; Granzymes; Hydroquinones; Ionomycin; Killer Cells, Natural; Lymphocyte Activation; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Perforin; Spleen

Most Ca(2+) signaling research in platelets has relied solely on monitoring the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). Changes in [Ca(2+)](cyt) constitute the net effect of Ca(2+) fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca(2+) sequestration into the stores and Ca(2+) removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca(2+) signaling difficult and subject to error.. To validate the use of the low-affinity Ca(2+) indicator Fluo-5N to monitor the concentration of Ca(2+) in the intracellular stores ([Ca(2+)](st)) of human platelets as a first step in developing assays for a systems-level analysis of platelet Ca(2+) signaling.. Fluo-5N-loaded and Fura-2-loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca(2+)](cyt) and [Ca(2+)](st).. Fluo-5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca(2+) stores and to physiologic agonists. Ca(2+) reuptake inhibitors and blockers of Ca(2+) release channels had the expected effects on Fura-2 and Fluo-5N fluorescence. Agonist-evoked Ca(2+) release was reversed by Ca(2+) addition to the medium, and required intact Ca(2+) reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca(2+) -ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non-SERCA Ca(2+) reuptake mechanism. Evidence for a role for Ca(2+) -induced Ca(2+) release in agonist-evoked responses was obtained.. Our data provide a validation of the use of Fluo-5N as a method for monitoring changes in [Ca(2+)](st) in human platelets.

Topics: Blood Platelets; Calcium; Calcium Signaling; Fluorescent Dyes; Fura-2; Humans; Hydroquinones; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Fluid; Ionomycin; Microscopy, Confocal; NADP; Nigericin; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Thrombin

Phasic myometrial contractions utilize mechanisms involving the cycling of calcium into and out of intracellular calcium stores. These studies were performed to determine the effects of 2,5-di(tert-butyl)-1,4-hydroquinone (tBHQ), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, on in vitro isometric myometrial contractions. These studies demonstrated that low concentrations of tBHQ (eg. 10 microM) appear to inhibit intracellular calcium cycling, whereas higher concentrations also inhibit extracellular calcium influx. These combined tBHQ effects markedly suppressed myometrial contractions stimulated in response to various agonists including oxytocin, PGF2 alpha, KCl, ionomycin, and Bay K 8644.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium-Transporting ATPases; Dinoprost; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Female; Hydroquinones; Ionomycin; Myometrium; Oxytocin; Potassium Chloride; Rats; Rats, Sprague-Dawley; Uterine Contraction

A close correlation was observed between intracellular Ca2+ pool depletion and refilling and the onset of DNA synthesis and proliferation of DDT1MF-2 smooth muscle cells. The intracellular Ca2+ pump inhibitors 2,5-di-tert-butyl-hydroquinone (DBHQ) and thapsigargin (TG) specifically emptied identical inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools and both arrested cell growth at concentrations corresponding to Ca2+ pump blockade. However, an important distinction was observed between the two inhibitors with respect to their reversibility of action. Upon removal of DBHQ from DBHQ-arrested cells, Ca2+ pools immediately refilled, and 14 hr later cells entered S phase followed by normal cell proliferation; the time for entry into S phase was identical to that for cells released from confluence arrest. Although TG irreversibly blocked Ca2+ pumping and emptied Ca2+ pools, high serum treatment of TG-arrested cells induced recovery of functional Ca2+ pools in 6 hr (via probable synthesis of new pump); thereafter cells proceeded to S phase and normal cell proliferation within the same time period (14 hr) as that following release of DBHQ-arrested cells. The precise relationship between Ca2+ pump blockade and growth arrest indicates that Ca2+ pool emptying maintains cells in a G0-like quiescent state; upon refilling of pools, normal progression into the cell cycle is resumed. It is possible that a specific cell cycle event necessary for G0 to G1 transition depends upon signals generated from the InsP3-sensitive Ca2+ pool.

Topics: Animals; Antioxidants; Calcium; Calcium-Transporting ATPases; Cell Division; Cell Line; Fura-2; Hydroquinones; Inositol 1,4,5-Trisphosphate; Ionomycin; Kinetics; Muscle, Smooth; Spectrometry, Fluorescence; Terpenes; Thapsigargin; Time Factors