We have shown that estrogens and calcitriol, the hormonally active form of vitamin D, increase the concentration of intracellular calcium ([Ca 2؉ ] i ) within 5 s by mobilizing calcium from the endoplasmic reticulum and the formation of inositol 1,4,5-trisphosphate and diacylglycerol. Because the activation of effectors as phospholipase C (PLC) coupled to G-proteins is the early event in the signal transduction pathway leading to the inositol 1,4,5-trisphosphate formation and to [Ca 2؉ ] i increase, we described different PLC isoforms (1, 2, ␥1, and ␥2, but not 4) in female rat osteoblasts using Western immunoblotting. The data showed that phospholipase C  was involved in the mobilization of Ca 2؉ from the endoplasmic reticulum of Fura-2-loaded confluent osteoblasts by calcitriol and 17 estradiol, and PLC ␥ was ineffective. The data also showed that only a PLC 1 linked to a Pertussis toxin-insensitive G-protein and a PLC 2 coupled to a Pertussis toxin-sensitive G-protein are involved in the effects of calcitriol and 17 estradiol on the mobilization of Ca 2؉ from intracellular Ca 2؉ stores. In conclusion, these results may be an important step toward understanding membrane effects of these steroids and may be an additional argument in favor of membrane receptors to steroid hormones.An increase in the turnover of inositol lipids in response to receptor is one of the most important molecular mechanisms used by cells for transmembrane signaling. The initial event is the hydrolysis of phosphatidylinositol 4,5-bisphosphate, a reaction catalyzed by a phosphoinositide-specific phospholipase C (PLC), 1 which generates two intracellular second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (1-4). Inositol 1,4,5-trisphosphate binds to specific receptors on the endoplasmic reticulum (5) and mobilizes intracellular calcium, whereas diacylglycerol activates protein kinase C (6), which results in increased phosphorylation of cellular proteins.Molecular cloning has revealed at least three major families of PLC, , ␥, and ␦ (7-9). Each of these families occurs in a number of isoforms. The enzymes are classified on the basis of their size and their immunological and structural similarities. The PLC isoforms have two highly conserved domains, X and Y, which form the active site of the protein. PLC ␦ and PLC ␥ proteins differ from PLC  in that they have shorter C-terminal extensions past the end of the Y domain (9). This diversity among the PLC isoforms also extends to distinct mechanisms of regulation and function for the three PLC families. PLC ␥ is regulated via the phosphorylation of tyrosine residues between the X and Y domains by receptor tyrosine kinases (10 -12). PLC  enzymes, of which there are four isoforms, PLC 1-4, are regulated via heterotrimeric G-proteins in response to an agonist binding to a receptor (13-15). The way in which PLC ␥ is regulated is not yet known, but enzyme activity is not affected by either the G-protein subunits or by receptor tyrosine kinases (16).The activat...
Progressive familial intrahepatic cholestasis type 2 (PFIC2) is a result of mutations in ABCB11 encoding bile salt export pump (BSEP), the canalicular bile salt export pump of hepatocyte. In some PFIC2 patients with missense mutations, BSEP is not detected at the canaliculus owing to mistrafficking of BSEP mutants. In vitro, chaperone drugs, such as 4‐phenylbutyrate (4‐PB), have been shown to partially correct mistrafficking. Four PFIC2 patients harboring at least one missense mutation (p.G982R, p.R1128C, and p.T1210P) were treated orally with 4‐PB and followed prospectively. Patient mutations were reproduced in a Bsep/green fluorescent protein plasmid. Cellular localization of the resulting Bsep mutants was studied in a hepatocellular line (Can 10), and effects of treatment with 4‐PB and/or ursodeoxycholic acid (UDCA) were assessed. In Can 10 cells, Bsep mutants were detected in the endoplasmic reticulum instead of at the canalicular membrane. Treatment with 4‐PB and UDCA partially corrected Bsep mutant targeting. With 4‐PB, we observed, in all patients, a decrease of pruritus and serum bile acid concentration (BAC) as well as an improvement of serum liver tests. Pathological liver injuries improved, and BSEP, which was not detected at the canalicular membrane before treatment, appeared at the canalicular membrane. Bile analyses showed an increase in BAC with 4‐PB. Patient conditions remained stable with a median follow‐up of 40 months (range, 3‐53), and treatment tolerance was good. Conclusion: 4‐PB therapy may be efficient in selected patients with PFIC2 owing to ABCB11 missense mutations affecting BSEP canalicular targeting. Bile secretion improvement may be a result of the ability of 4‐PB to retarget mutated BSEP. (Hepatology 2015) Hepatology 2015;62:558–566
The correct functioning of the liver is ensured by the setting and the maintenance of hepatocyte polarity. The complex polarity of the hepatocyte is characterized by the existence of several basolateral and apical poles per cell. Many in vitro models are available for studying hepatocyte polarity, but which are the more suitable? To answer this question, we aimed to identify criteria which determine the typical hepatocyte polarity. Therefore, we compiled a range of protein markers of membrane domains in rat hepatocytes and investigated their involvement in hepatocytic functions. Then, we focused on the relationship between hepatic functions and the cytoskeleton, Golgi apparatus and endoplasmic reticulum. Subsequently, we compared different cell lines expressing hepatocyte polarity. Finally, to demonstrate the usefulness of some of these lines, we presented new data on endoplasmic reticulum organization in relation to polarity.
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