Changes in expression of connexin 32, bile canaliculus-like structures, and localization of alkaline phosphatase in primary cultures of fetal rat hepatocytes.

We devised an experimental design in primary cultures of fetal rat hepatocytes for studying hepatocyte differentiation over a short period. In the present study, hepatocytes were first cultured for 3 days in dexamethasone-supplemented medium and then for an additional 3 days in dexamethasone- or epidermal growth factor-supplemented medium. In hepatocytes cultured continuously in dexamethasone-supplemented medium, the expression of connexin 32 increased and bile canaliculus-like structures and localization of alkaline phosphatase in the plasma membrane around bile canaliculus-like structures were maintained. Few cells incorporated bromodeoxyuridine. On the other hand, in most of the hepatocytes cultured in epidermal growth factor-supplemented medium, the expression of connexin 32 was minimally recognized, bile canaliculus-like structures were shortened or eliminated, and alkaline phosphatase was localized as numerous fine spots throughout the cytoplasm. More than 20% of all hepatocytes incorporated bromodeoxyuridine. The present study suggests that in hepatocytes, there is a close relationship among connexin 32 expression, the maintenance of bile canaliculus-like structures, and the localization of alkaline phosphatase to the plasma membrane around the bile canaliculus-like structures, and this indicates that the present experimental model is useful for studying hepatocyte differentiation over a short period.

Semi-automatic counting of connexin 32s immunolocalized in cultured fetal rat hepatocytes using image processing.

Connexin 32s (Cx32s) were immunolocalized in fetal rat hepatocytes and their distribution was determined qualitatively. We used image analysis using a quantitative index (QI) of Cx32 (QI Cx32) defined as the area of Cx32s/number of cells in cultured fetal rat hepatocytes. Hepatocytes from livers of fetal rats were separated by collagenase digestion and low centrifugation on gestational day 17. Cells were cultured for 3 days in dexamethasone (DEX)-supplemented medium (Dex0). The medium was replaced with fresh medium and cells were continuously cultured for 3 days with DEX or epidermal growth factor supplemented medium (Dex3 or EGF3). After culture termination, cells were fixed and stained using the fluorescein-labeled antibody method for Cx32s and diaminophenylindole staining for nuclei. Thirty pairs of histological images for Cx32s and nuclei, 180 images in total, were obtained from each condition. The QI Cx32 significantly increased from 284.1 ± 102.0 (mean and SD, n=26) of Dex0 to 428.9 ± 101.0 of Dex3 (n=28) (P<0.05, Kruskal-Wallis test, then Steel-Dwass test). The increase of QI Cx32 was compatible with the morphological observations. The image analysis processing time after preparation for 180 images was reduced from 8h needed for manual operations to 1 min using ImageJ software with our macro routine.

Localization of Alkaline Phosphatase and Cathepsin D during Cell Restoration after Colchicine Treatment in Primary Cultures of Fetal Rat Hepatocytes.

Localization of alkaline phosphatase (ALP) and cathepsin D (CAPD) in primary cultures of fetal rat hepatocytes was examined using double immunofluorescent staining in order to investigate the relationship between lysosome movement and the fate of ALP during cell restoration after microtubule disruption by colchicine. At 3 hr and 24 hr after colchicine treatment, numerous coarse dots containing ALP were observed throughout the cytoplasm, and some of these showed colocalization with CAPD. At 48 hr and 72 hr after colchicine treatment, although most of the dots containing ALP in the cytoplasm disappeared, dots containing CAPD remained. The present results suggest that the denatured ALP proteins remaining in the cytoplasm of hepatocytes during cell restoration after colchicine treatment are digested by lysosomes.

Change in localization of alkaline phosphatase and mannosidase II by colchicine treatment of primary cultures of fetal rat hepatocytes.

We examined the changes in localization of alkaline phosphatase (ALP) and mannosidase II (man II), a Golgi marker, after colchicine treatment of primary cultures of fetal rat hepatocytes, using double immunofluorescence staining and confocal laser microscopy. In hepatocytes cultured in basal medium, ALP was localized in the perinuclear cytoplasm, and man II was observed in the Golgi region of the cytoplasm. When hepatocytes were cultured in dexamethasone-supplemented medium, ALP was also localized in the plasma membrane surrounding the bile canaliculus-like structure that was formed between adjacent cells. In hepatocytes cultured in the same medium containing colchicine, the structure of microtubules in the cytoplasm was lost, man II exhibited granular distribution scattering throughout the cytoplasm, and ALP was localized in coarse granular sites of the cytoplasm. However, ALP was not colocalized at the same sites as man II. The present study indicated that colchicine inhibits the dexamethasone-promoted translocation of ALP to the plasma membrane surrounding the bile canaliculus-like structure in primary cultures of fetal rat hepatocytes by disassembling microtubules and discomposing the Golgi complex.

Effects of colchicine on localization of alkaline phosphatase in McA-RH 7777 rat hepatoma cells.

We investigated the changes caused by microtubule disruption in cell contact-induced translocation of alkaline phosphatase (ALP) from the Golgi area to the plasma membrane in McA-RH 7777 cells. When the cells were treated with colchicine, the tubular structure of microtubules in the cytoplasm was lost. Colchicine treatment also resulted in the appearance of numerous dots containing mannosidase II (man II) throughout the cytoplasm. Moreover, ALP was distributed in small dots throughout the cytoplasm, as well as in all regions of the plasma membrane, although it was most concentrated at sites of intercellular contact. On the other hand, when the cells were incubated in basal medium after colchicine treatment, large spots containing ALP reappeared in the perinuclear cytoplasm more quickly than the accumulation of small dots containing man II. These findings suggest that colchicine causes disassembly of the Golgi complex into fragments, which scatter throughout the cytoplasm, but that it does not interfere with translocation of ALP to the plasma membrane. Furthermore, cytoplasmic ALP may be localized at sites other than the Golgi complex.

Localization of alkaline phosphatase and proteins related to intercellular junctions in primary cultures of fetal rat hepatocytes.

The localization of alkaline phosphatase (ALP) and four proteins related to intercellular junctions in primary cultures of fetal rat hepatocytes was immunocytochemically investigated using fluorescence-labeled antibodies and confocal laser microscopy in order to determine whether the formation of intercellular junctions at the borders between adjacent rat hepatocytes becomes the trigger of translocation of ALP from the cytoplasm to the plasma membrane. Dexamethasone (DEX) which was supplemented in the base medium promoted the translocation of ALP from the cytoplasm to the plasma membrane surrounding bile canaliculus-like intercellular spaces and the appearance of connexin-32 at cell borders between adjacent fetal hepatocytes. E-cadherin, occludin and ZO-1 were localized at the cell borders between adjacent fetal hepatocytes irrespective of the presence of DEX. Occludin and ZO-1 were further localized along the plasma membrane surrounding bile canaliculus-like intercellular spaces formed by DEX. The present study indicates that the formation of adherens and tight junctions between adjacent rat hepatocytes does not become the trigger of ALP translocation from the cytoplasm to the plasma membrane, although we cannot be certain of whether the formation of gap junctions between adjacent rat hepatocytes triggers ALP translocation.

Localization of alkaline phosphatase and proteins related to intercellular junctions in rat hepatoma cell line McA-RH 7777.

We investigated the localization of alkaline phosphatase (ALP) and three proteins related to intercellular junctions in the McA-RH 7777 rat hepatoma cell line to determine if the formation of junctions between adjacent McA-RH 7777 cells triggers translocation of ALP from cytoplasm to the plasma membrane. Contact between adjacent McA-RH 7777 cells promotes translocation of ALP from the Golgi area of the cytoplasm to the plasma membrane, and also promotes translocation of two proteins, E-cadherin and ZO-1, related to intercellular junctions, from cytoplasm to the plasma membrane.

Computer reconstruction of the three-dimensional structure of mouse cerebral ventricles.

In the brain, the cerebral ventricles are important as the site of cerebrospinal fluid production. The cerebral ventricles consist of the lateral ventricles, the third ventricle and the fourth ventricle, and their stereoscopic structures and the connections between them are not visible from outside of the brain. Observation of the stereoscopic structure of cerebral ventricles is possible by producing a mold preparation of the ventricular system [Systematic Human Anatomy (1984) 589; A Colour Atlas of Human Anatomy (1993) 79; J. Anat. 68 (1934) 480; Brain 75 (1952) 259]. However, this method does not facilitate visualization of the position of the ventricles within the brain or the positional relationships between the cerebral ventricles and other structures. Murine brains are often used in studies of hydrocephalus [Mol. Cell. Biol. 22 (2002) 2769; Brain Res. 891 (2001) 247]. Rolf et al. have reported that a few L1 mutant mice display severe hydrocephalus and suggested that, in such mice, massively enlarged ventricles create deformations of the brain, which secondarily cause stenosis of the aqueducts followed by severe hydrocephalus [Brain Res. 891 (2001) 247]. The three-dimensional structure of murine cerebral ventricles reconstructed on a computer display would be extremely useful not only for stereoscopic observation of the cerebral ventricles in murine brains but also in morphological analysis of murine cerebral ventricles using additional software for three-dimensional measurement. We therefore attempted a three-dimensional computer reconstruction of the cerebral ventricles from serial histological sections of whole mouse brain in order to facilitate research into hydrocephalus using mouse models. In this study, mouse brain is fixed in formalin and then embedded in paraffin after immersion in celloidin. Serial histological sections are produced and digitized, and outlines of the brain surface and cerebral ventricular faces are traced on printouts. Serial drawings are again digitized, and the three-dimensional structure of the brain surface and cerebral ventricles is reconstructed on the computer using reconstruction software. Using this technique, we accurately described the mouse brain surface and cerebral ventricle faces on a computer display.