Doxorubicin induces EGF receptor-dependent downregulation of gap junctional intercellular communication in rat liver epithelial cells

Exposure of rat liver epithelial cells to doxorubicin, an anthraquinone derivative widely employed in cancer chemotherapy, led to a dose-dependent decrease in gap junctional intercellular communication (GJC). Gap junctions are clusters of inter-cellular channels consisting of connexins, the major connexin in the cells used being connexin-43 (Cx43). Doxorubicin-induced loss of GJC was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated using inhibitors of ERK activation. Furthermore, activation of the epidermal growth factor (EGF) receptor by doxorubicin was responsible for ERK activation and the subsequent attenuation of GJC. Inhibition of GJC, however, was not by direct phosphorylation of Cx43 by ERK-1/2, whereas menadione, a 1,4-naphthoquinone derivative that was previously demonstrated to activate the same EGF receptor-dependent pathway as doxorubicin, resulting in downregulation of GJC, caused strong phos-phorylation of Cx43 at serines 279 and 282. Thus, ERK-dependent downregulation of GJC upon exposure to quinones may occur both by direct phosphorylation of Cx43 and in a phosphorylation-independent manner.     

Spatial learning induces predominant downregulation of cytosolic proteins in the rat hippocampus

Spatial learning is known to depend on protein synthesis in the hippocampus. Whereas the role of the hippocampus in spatial memory is established, the biochemical and molecular mechanisms underlying this process are poorly understood. To comprehend the complex pattern of protein expression induced by spatial learning, we analyzed alterations in the rat hippocampus proteome after 7 days of spatial learning in the Morris water maze. Forty Wistar rats were randomized into two groups. Animals of group A learned to localize a hidden platform in the water maze. Animals of group B served as controls and spent exactly the same time in the water maze as animals of group A. However, no platform was used in this test and the rats could not learn to localize the target. After the last trial, hydrophilic proteins from the hippocampus were isolated. A proteome-wide study was performed, based on two-dimensional gel electrophoresis and mass spectrometry. Compared with non-learning animals, 53 (70%) proteins were downregulated and 23 (30%) proteins were upregulated after 7 days in rats with spatial learning. The overall changes in protein expression, as quantified by the induction factor, ranged from -1.62 (downregulation to 62%) to 2.10 (upregulation by 110%) compared with controls (100%). Most identified proteins exhibit known functions in vesicle transport, cytoskeletal architecture, and metabolism as well as neurogenesis. These findings indicate that learning in the Morris water maze has a morphological correlate on the proteome level in the hippocampus.     

Regeneration of white rat liver during cholestasis

Cholestasis inhibits expression of early response genes in rat hepatocytes stimulated to proliferation and restricts the process related to reparative regeneration. The occlusion of general bile duct induces bile acid accumulation, which suppresses 11-beta-hydroxysteroid-dehydrogenase activity. Hence, the concentration of corticosterone is increased, which suppresses the expression of early response genes in rat hepatocytes. The restoration of liver mass occurs due to proliferation of reserved cells. An unusual mitotic peak of these cells is registered 34 h after the operation.     

Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract

A remarkable amount, of water is transported in the gastrointestinal (GI) organs to fulfil the secretory and absorptive functions of the GI tract. However, the molecular basis of water movement in the GI epithelial barriers is still poorly known. Important clues about the mechanisms by which water is transported in the GI tract were provided by the recent identification of multiple aquaporin water channels expressed in GI tissues. Here we define the mRNA and protein expression and the cellular and subcellular distribution of aquaporin-8 (AQP8) in the rat GI tract. By semi-quantitative RT-PCR the AQP8 mRNA was detected in duodenum, proximal jejunum, proximal colon, rectum, pancreas and liver and, to a lesser extent, in stomach and distal colon. Immunohistochemistry using affinity-purified antibodies revealed AQP8 staining in the absorptive epithelial cells of duodenum, proximal jejunum, proximal colon and rectum where labeling was largely intracellular and confined to the subapical cytoplasm. Confirming previous results, AQP8 staining was seen at the apical pole of pancreatic acinar cells. Interestingly, both light and immunoelectron microscopy analyses showed AQP8 reactivity in liver where labeling was associated to hepatocyte intracellular vesicles and over the plasma membrane delimiting the bile canaliculi. A complex pattern was observed by immunoblotting with total membranes of the above GI organs incubated with affinity-purified anti-AQP8 antibodies which revealed multiple bands with molecular masses ranging between 28 and 45 kDa. This immunoblotting pattern was not modified after deglycosylation with N-glycosidase F except the 34-kDa band of liver that, as already reported, was partially down-shifted to 28 kDa. No bands were detected after preadsorption of the anti-AQP8 antibodies with the immunizing peptide. The cellular and subcellular distribution of AQP8 suggest physiological roles for this aquaporin in the absorption of water in the intestine and the secretion of bile and pancreatic juice in liver and pancreas, respectively. The large intracellular expression of AQP8 may indicate its recycling between the cytoplasmic compartment and the plasma membrane. The cytoplasmic localization observed may also relate to the involvement of AQP8 in processes of intracellular osmoregulation.     

Knockdown of aquaporin-8 induces mitochondrial dysfunction in 3T3-L1 cells

BackgroundAquaporin-8 (AQP8), a member of the aquaporin water channel family, is expressed in various tissue and cells, including liver, testis, and pancreas. AQP8 appears to have functions on the plasma membrane and/or on the mitochondrial inner membrane. Mitochondrial AQP8 with permeability for water, H₂O₂ and NH₃ has been expected to have important role in various cells, but its information is limited to a few tissues and cells including liver and kidney. In the present study, we found that AQP8 was expressed in the mitochondria in mouse adipose tissues and 3T3-L1 preadipocytes, and investigated its role by suppressing its gene expression.MethodsAQP8-knocked down (shAQP8) cells were established using a vector expressing short hairpin RNA. Cellular localization of AQP8 was examined by western blotting and immunocytochemistry. Mitochondrial function was assessed by measuring mitochondrial membrane potential, oxygen consumption and ATP level measurements.ResultsIn 3T3-L1 cells, AQP8 was expressed in the mitochondria. In shAQP8 cells, mRNA and protein levels of AQP8 were decreased by about 75%. The shAQP8 showed reduced activities of complex IV and ATP synthase; it is probable that the impaired mitochondrial water handling in shAQP8 caused suppression of the electron transport and ADP phosphorylation through inhibition of the two steps which yield water. The reduced activities of the last two steps of oxidative phosphorylation in shAQP8 cause low routine and maximum capacity of respiration and mitochondrial hyperpolarization.ConclusionMitochondrial AQP8 contributes to mitochondrial respiratory function probably through maintenance of water homeostasis.General significanceThe AQP8-knocked down cells we established provides a model system for the studies on the relationships between water homeostasis and mitochondrial function.     

Diabetes induces metabolic adaptations in rat liver mitochondria: role of coenzyme Q and cardiolipin contents

Several studies have been carried out to evaluate the alterations in mitochondrial functions of diabetic rats. However, results are sometimes controversial, since experimental conditions diverge, including age and strain of used animals. The purpose of this study was to evaluate the metabolic modifications in liver mitochondria, both in the presence of severe (STZ-treated rats) and mild hyperglycaemia [Goto-Kakizaki (GK) rats], when compared with control animals of similar age. Moreover, metabolic alterations were evaluated also at initial and advanced stages of the disease. We observed that both models of diabetes (type 1 and type 2) presented a decreased susceptibility of liver mitochondria to the induction of permeability transition (MPT). Apparently, there is a positive correlation between the severity of diabetes mellitus (and duration of the disease) and the decline in the susceptibility to MPT induction. We also found that liver mitochondria isolated from diabetic rats presented some metabolic adaptations, such as an increase in coenzyme Q and cardiolipin contents, that can be responsible for the observed decrease in the susceptibility to multiprotein pore (MPTP) opening.     

miR-214-mediated downregulation of RNF8 induces chromosomal instability in ovarian cancer cells

Defective DNA damage response (DDR) is frequently associated with carcinogenesis. Abrogation of DDR leads to chromosomal instability, a most common characteristic of tumors. However, the molecular mechanisms underlying regulation of DDR are still elusive. The ubiquitin ligase RNF8 mediates the ubiquitination of γH2AX and recruits 53BP1 and BRCA1 to DNA damage sites which promotes DDR and inhibits chromosomal instability. Though RNF8 is a key player involved in DDR, regulation of its expression is still poorly understood. Here, we show that miR-214 could abrogate DDR by repressing RNF8 expression through direct binding to 3′-untranslated region (3′ UTR) of RNF8 mRNA in human ovarian cancer cells. Antagonizing miR-214 by expressing its inhibitors in A2780 cells significantly increased RNF8 expression and thus promoted DNA damage repair. Consistent with the role of miR-214 in regulating RNF8 expression, the impaired DNA repair induced by miR-214 overexpression can be rescued by overexpressing RNF8 mRNA lacking the 3′ UTR. Together, our results indicate that down-regulation of RNF8 mediated by miR-214 impedes DNA damage response to induce chromosomal instability in ovarian cancers, which may facilitate the understanding of mechanisms underlying chromosomal instability.     

Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.     

Adrenaline induces mitochondrial biogenesis in rat liver

We studied the effects of adrenaline administration and depletion (induced by reserpine) on rat liver oxidative metabolism. We showed that adrenaline increases, and reserpine decreases aerobic capacity (inferred by cytochrome oxidase activity) in tissue modifying the hepatic content of mitochondrial proteins without changing mitochondrial aerobic capacity. The changes in tissue cytochrome oxidase activity, which agreed with the expression levels of factors involved in mitochondrial biogenesis, such as PGC-1, NRF-1, and NRF-2, were associated with similar changes in tissue and mitochondrial State 3 respiration. Adrenaline and reserpine induced extensive lipid and protein oxidative damage in tissue and mitochondria. The increase in H₂O₂ release by respiring mitochondria and the decrease in the activities of the antioxidant enzymes glutathione peroxidase and reductase contributed to the reserpine effect on oxidative damage. The adrenaline effect is more difficult to explain, since the hormone increased the antioxidant enzyme activities but, in respiring mitochondria, increased ROS release rate in the presence of succinate and decreased it in the presence of pyruvate/malate. These opposite changes were due to the increased content of the autoxidizable electron carrier located at complex III and decreased content of that located at complex I. Our data suggest that adrenaline can be involved in the mitochondrial population adaptation which verify in conditions in which an increased body energy expenditure verify such as cold exposure.     

Interleukin-1beta induces the expression of aquaporin-4 through a nuclear factor-kappaB pathway in rat astrocytes

Interleukin (IL)-1beta is known to play a role in the formation of brain edema after various types of injury. Aquaporin (AQP)4 is also reported to be involved in the progression of brain edema. We tested the hypothesis that AQP4 is induced in response to IL-1beta. We found that expression of AQP4 mRNA and protein was significantly up-regulated by IL-1beta in cultured rat astrocytes, and that intracerebroventricular administration of IL-1beta increased the expression of AQP4 protein in rat brain. The effects of IL-1beta on induction of AQP4 were concentration and time dependent. The effects of IL-1beta on AQP4 were mediated through IL-1beta receptors because they were abolished by co-incubation with IL-1 receptor antagonist. It appeared that IL-1beta increased the level of AQP4 mRNA without involvement of de novo protein synthesis because cycloheximide, a protein synthesis inhibitor, did not inhibit the effects of IL-1beta. Inhibition of the nuclear factor-kappaB (NF-kappaB) pathway blocked the induction of AQP4 by IL-1beta in a concentration-dependent manner. These findings show that IL-1beta induces expression of AQP4 through a NF-kappaB pathway without involvement of de novo protein synthesis in rat astrocytes.     

Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% (P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (-31%, P < 0.05) as well as in cells exposed to acid (-90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH(4)Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.     

Polyploidization in rat liver: the role of binucleate cells.

Liver cells were isolated from rats undergoing active formation of tetraploid cells and prelabelled in their DNA with 14C thymidine. Autoradiography of the isolated cells showed that binucleate diploid cells, a major component of the parenchymal cell population at this time, are also active in DNA synthesis. These cells probably pass through mitosis and generate mononucleate tetraploid cells, the dominant cell type of mature rat liver, since the frequency of binucleate tetraploid cells is very low at this stage in rat liver development. The biological significance of liver polyploidy is discussed and it is suggested that this lies in enhanced resistance to mutagenesis.     

Mirex induces ornithine decarboxylase in female rat liver

Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, was significantly induced in female rat liver following oral administration of the pesticide mirex. After dual oral exposure (120 mg/kg of mirex; 21 and 4 hr prior to sacrifice), ornithine decarboxylase activity in rat liver cytosol was 70-fold higher than control values. A single oral dose of mirex (180 mg/kg) induced hepatic ornithine decarboxylase activity 55-fold over controls. After a single oral dose of mirex the maximal induction of ODC activity occurred at 36 hr. Mirex is an unusually potent and long-lasting inducer of rat hepatic ornithine decarboxylase activity.     

Maternal LPS induces cytokines in the amniotic fluid and corticotropin releasing hormone in the fetal rat brain

Perinatal infections are a risk factor for fetal neurological pathologies, including cerebral palsy and schizophrenia. Cytokines that are produced as part of the inflammatory response are proposed to partially mediate the neurological injury. This study investigated the effects of intraperitoneal injections of lipopolysaccharide (LPS) to pregnant rats on the production of cytokines and stress markers in the fetal environment. Gestation day 18 pregnant rats were treated with LPS (100 microg/kg body wt i.p.), and maternal serum, amniotic fluid, placenta, chorioamnion, and fetal brain were harvested at 1, 6, 12, and 24 h posttreatment to assay for LPS-induced changes in cytokine protein (ELISA) and mRNA (real-time RT-PCR) levels. We observed induction of proinflammatory cytokines interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha (TNF-alpha) as well as the anti-inflammatory cytokine IL-10 in the maternal serum within 6 h of LPS exposure. Similarly, proinflammatory cytokines were induced in the amniotic fluid in response to LPS; however, no significant induction of IL-10 was observed in the amniotic fluid. LPS-induced mRNA changes included upregulation of the stress-related peptide corticotropin-releasing factor in the fetal whole brain, TNF-alpha, IL-6, and IL-10 in the chorioamnion, and TNF-alpha, IL-1 beta, and IL-6 in the placenta. These findings suggest that maternal infections may lead to an unbalanced inflammatory reaction in the fetal environment that activates the fetal stress axis.