The Reduced Expression of 6ckine in the plt Mouse Results from the Deletion of One of Two 6ckine Genes

6Ckine is an unusual chemokine capable of attracting naive T lymphocytes in vitro. It has been recently reported that lack of 6Ckine expression in lymphoid organs is a prominent characteristic of mice homozygous for the paucity of lymph node T cell (plt) mutation. These mice show reduced numbers of T cells in lymph nodes, Peyer's patches, and the white pulp of the spleen. The genetic reason for the lack of 6Ckine expression in the plt mouse, however, has remained unknown. Here we demonstrate that mouse 6Ckine is encoded by two genes, one of which is expressed in lymphoid organs and is deleted in plt mice. A second 6Ckine gene is intact and expressed in the plt mouse

Hydrophobic bile salts inhibit gallbladder smooth muscle function via stimulation of GPBAR1 receptors and activation of KATP channels

Hydrophobic bile salts are thought to contribute to the disruption of gallbladder smooth muscle (GBSM) function that occurs in gallstone disease, but their mechanism of action is unknown. The current study was undertaken to determine how hydrophobic bile salts interact with GBSM, and how they reduce GBSM activity. The effect of hydrophobic bile salts on the activity of GBSM was measured by intracellular recording and calcium imaging using wholemount preparations from guinea pig and mouse gallbladder. RT-PCR and immunohistochemistry were used to evaluate expression of the G protein-coupled bile acid receptor, GPBAR1. Application of tauro-chenodeoxycholate (CDC, 50–100 μm) to in situ GBSM rapidly reduced spontaneous Ca2+ flashes and action potentials, and caused a membrane hyperpolarization. Immunoreactivity and transcript for GPBAR1 were detected in gallbladder muscularis. The GPBAR1 agonist, tauro-lithocholic acid (LCA, 10 μm) mimicked the effect of CDC on GBSM. The actions of LCA were blocked by the protein kinase A (PKA) inhibitor, KT5720 (0.5–1.0 μm) and the KATP channel blocker, glibenclamide (10 μm). Furthermore, LCA failed to disrupt GBSM activity in Gpbar1−/− mice. The findings of this study indicate that hydrophobic bile salts activate GPBAR1 on GBSM, and this leads to activation of the cyclic AMP–PKA pathway, and ultimately the opening of KATP channels, thus hyperpolarizing the membrane and decreasing GBSM activity. This inhibitory effect of hydrophobic bile salt activation of GPBAR1 could be a contributing factor in the manifestation of gallstone disease

Resveratrol Affects Sphingolipid Metabolism in A549 Lung Adenocarcinoma Cells

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity—apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)—in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies