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BACKGROUND: Factors influencing parasitosis in cattle in Bangladesh remain inadequately explored, necessitating a comprehensive investigation for interventions and sustainable livestock farming. OBJECTIVES: We conducted this study to estimate the prevalence and distribution of gastrointestinal parasites, exploring their intricate relationship with farm management practices across a spectrum of small-, medium-, and large-scale commercial farms. METHODS: We conducted this study in the Chattogram district of Bangladesh. We collected a total of 189 freshly voided faecal samples from different farms. We recorded the age, breed, milking status, sex, body condition score, and anthelmintic use history of the sampled animals. We processed the samples using the direct smear method, with the identification of one egg per sample being considered positive. RESULTS: We estimated the prevalence of gastrointestinal parasite infection in large-scale (52.1%), medium-scale (54.5%), and small-scale farms (70.0%), with statistically significant differences (p ≤ 0.05). Both pregnant and lactating cows, as well as indigenous cattle, were more likely to have gastrointestinal parasites (p ≤ 0.05). The predominant parasites across farms of all sizes were trematodes (Paramphistomum spp. and Schistosomas spp.) and protozoa (Balantidium coli and Coccidia spp.). CONCLUSION: Poor farm management practices, such as no pasture management and inadequate deworming regimens, may contribute to the elevated prevalence and infection load observed on small-scale farms. The increased parasitosis in previously dewormed animals can be attributed to the development of anthelmintic resistance against gastrointestinal parasites. Implementing proper and effective deworming strategies is crucial to preventing gastrointestinal parasitosis and mitigating the risk of anthelmintic resistance.

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Work-based learning (WBL) provides relevant contemporary experience of working environments. Potential benefits for students include developing invaluable skills (clinical, personal, cultural, and professional) and gaining greater awareness of the profession and future career opportunities. However, there are also challenges related to running and sustaining a successful WBL program. In the context of this study, WBL refers to external placements undertaken by final-year students. The aims of the study were to identify ways to optimize the benefits while managing the challenges in delivering WBL in a veterinary curriculum. An in-depth study was undertaken at Chattogram Veterinary and Animal Sciences University (CVASU), Bangladesh, where a WBL program has been in place for 20 years. Final-year veterinary students at CVASU were surveyed to ascertain WBL experiences; survey findings were further explored in focus groups with students, recent graduates, faculty, and placement providers. Most agreed that they had sufficient opportunities to observe, assist, and directly handle pet and farm animals with top skills learned, including clinical diagnosis and communication, and recognized the value of learning in professional workplaces. Based on suggested areas of improvement, the following recommendations can be made: carefully selecting placements, adjusting time allocation, improving communication and building strong collaborations with placement providers, allowing students to customize more placements to align with their career preferences, and staffing adequately to arrange placements and manage a WBL program. Overall, results suggest the current WBL arrangements at CVASU are reasonably good, but there are some specific areas for improvement.

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The World Health Organization declared coronavirus disease 2019 (COVID-19) a pandemic on March 11, 2020. COVID-19, the current global health emergency, is wreaking havoc on human health systems and, to a lesser degree, on animals globally. The outbreak has continued since the first report of COVID-19 in China in December 2019, and the second and third waves of the outbreak have already begun in several countries. COVID-19 is expected to have adverse effects on crop production, food security, integrated pest control, tourism, the car industry, and other sectors of the global economy. COVID-19 induces a range of effects in livestock that is reflected economically since human health and livelihood are intertwined with animal health. We summarize the potentially harmful effects of COVID-19 on livestock and possible mitigation steps in response to this global outbreak. Mitigation of the negative effects of COVID-19 and future pandemics on livestock requires the implementation of current guidelines.

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Antibiotic-free broiler meat production is becoming increasingly popular worldwide due to consumer perception that it is superior to conventional broiler meat. Globally, broiler farming impacts the income generation of low-income households, helping to alleviate poverty and secure food in the countryside and in semi-municipal societies. For decades, antibiotics have been utilized in the poultry industry to prevent and treat diseases and promote growth. This practice contributes to the development of drug-resistant bacteria in livestock, including poultry, and humans through the food chain, posing a global public health threat. Additionally, consumer demand for antibiotic-free broiler meat is increasing. However, there are many challenges that need to be overcome by adopting suitable strategies to produce antibiotic-free broiler meat with regards to food safety and chicken welfare issues. Herein, we focus on the importance and current scenario of antibiotic use, prospects, and challenges in the production of sustainable antibiotic-free broiler meat, emphasizing broiler farming in the context of Bangladesh. Moreover, we also discuss the need for and challenges of antibiotic alternatives and provide a future outlook for antibiotic-free broiler meat production.

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A cross-sectional observation and an intervention study were conducted in Chittagong, Bangladesh in 2015 to assess the status of antimicrobial residues in chicken and fish. The samples were tested for selected antimicrobials (amoxicillin, ciprofloxacin, oxytetracycline and enrofloxacin) using thin-layer chromatography (TLC). The TLC-based overall prevalence of residues was 87.9% in chicken (N = 182) and 56.9% in fish (N = 153). The prevalences in chicken in June (N = 91) and in October (N = 91), respectively, were 91.2% and 83.5% (amoxicillin), 1.1% and 1.1% (enrofloxacin), 1.1% and 0% (ciprofloxacin), and 0% and 6.6% (oxytetracycline). In fish, the prevalence in September (N = 74) and in October (N = 79) was 52.7% and 44.3% (amoxicillin) and 1.4% and 27.8% (oxytetracycline), respectively. The mean concentration of amoxicillin residue was evaluated by ultra-high-performance liquid chromatography to be 508.4 mg/kg (chicken) and 515.4 mg/kg (fish). The random effect model identified market (Chawkbazar vs. Boalkhali: OR 5.7; Steelmill bazar vs. Boalkhali: OR 5.6) as significant factors for amoxicillin residue in chicken. Amoxicillin concentration was significantly reduced in chicken of Kazirhat (ß= - 1.3) and Chawkbazar (ß= - 1.1) and increased in October (ß= 0.77) based on a generalized linear model (GLM). Climbing perch fish had significantly more risk of having amoxicillin residue than that of Bombay duck (OR = 0.05). All samples were treated by washing, boiling and cooking with spices, and then, TLC-based screening of amoxicillin residues was done. A subset of each treated group was evaluated by UHPLC. Treatment reduced amoxicillin residue levels significantly.

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Bangladesh experiences some of the highest malnutrition rates in the world, and efforts are being made to increase food security and overall health status. One of the largest constrains on increasing food security is endemic diseases among livestock and poultry populations. Newcastle Disease (ND) is one of these viral endemic diseases reducing food security. However, the sero- and viro-prevalence of ND has not been thoroughly studied in rural poultry in Bangladesh. Knowledge of farm management practices and their effect on ND sero and viro-prevalence is needed before interventions can occur, and efforts to improve the endemic state of ND cannot begin without a baseline study. This cross-sectional study randomly sampled 129 rural households with 245 chickens for the sero-prevalence and active infection rate of rural chickens in two selected upazilas (sub-districts) of the Chittagong district. ELISA was used for the detection of sero-prevalence, and cloacal samples were analyzed for ND presence using one-step RT-PCR. The aims of this study were to describe farmer demography, determine the ND sero-prevalence at the household and individual chicken level, estimate the proportionate ND prevalence at the individual chicken level, determine potential risk factors for ND sero-prevalence at the household level, and determine challenges farmers face with household chicken farming. The overall household level ND sero-prevalence based on ELISA was 31.8% (41/129) (95% CI: 23.9-40.6%), whereas the overall bird level ND sero-prevalence based on ELISA was 21.2% (52/245) (95% CI: 16.5-26.8%). ND prevalence based on RT-PCR was 12.5% (4/32) (95% CI: 3.5-29.0%). The odds of ND sero-positivity was significantly higher in farms belonging to Rangunia than in farms belonging to Anowara with an odds ratio (OR) of 7.8 (95% CI: 3.3-18.6%). The odds of ND sero-positivity was significantly lower in poultry house cleaning frequency of once or twice weekly compared with once daily cleaning (OR = 0.3; 95% CI: 0.1-0.8%). High cleaning frequency may produce excessive stress on poultry predisposing them to infection. Poultry rearing is different between Anowara and Rangunia. Anowara (coastal) scavenging areas become restricted because of regular tide flow allowing small fishes and other aquatic animals to be the dominant scavengers in Anowara. The incoming tide also removes viral reservoirs such as feces and dead birds that may otherwise be readily accessed by healthy chickens.

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Live Bird Markets (LBMs) in Asian countries are considered as hubs for the spread and maintenance of different infectious diseases. In Bangladesh, LBMs are the major source of live and dressed poultry to consumers and until now only a few studies have been conducted targeting infectious agent status such as avian influenza virus (AIV) prevalence of LBMs in Bangladesh. Therefore, a cross sectional study was conducted using all 40 LBMs within the Chittagong Metropolitan Area (CMA) of Bangladesh targeting demographic information and hygienic status of LBMs in concurrence with AIV prevalence and its subtype distribution, as well as the associated risk factors for AIV. Pooled environmental swab samples were collected from 2 to 9 different sites per stall, with epidemiological data being obtained from a total of 290 stalls across 40 LBMs. The samples were evaluated by Real Time Reverse Transcriptase Polymerase Chain Reaction. The prevalence of AIV was 40% (95% CI: 20-60%; N=40) at a LBM level followed by 20.3% (CI: 10-30%, N=290) at a stall level. Specifically, the prevalence of H5, H7 and H9 subtypes at stall level were 2.8% (95% CI: 1-5%), 0% (CI: 0-1.3%) and 3.1% (CI: 1-6%), respectively. Generalized Estimating Equation model identified that the type of species sold (OR=2.5: Chicken and non-duck species versus Duck with other species), bird holding areas (OR=1.9: Cage versus Floor) and Hygienic score (OR=3.1: Score 3 or more versus score less than 3) as potential risk factors for the detection of AIV at stall level. These results suggest that housing chickens and ducks together in the stalls, birds kept on floors, and lack of adequate hygienic measures of the stall were the crucial factors for spreading AIV. This research outcome could be used to develop a proof-based program concerning environmental sanitation along with development of an effective surveillance system to reduce the AIV transmission through LBMs in Bangladesh.

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The hepatotoxic bile acid glycochenodeoxycholate (GCDC) modulates hepatocyte cell death through activation of JNK, Akt, and Erk. The nonhepatotoxic bile acid taurocholate activates Akt and Erk through the sphingosine-1-phosphate receptor 2 (S1PR2). The role of the S1PR2 in GCDC-mediated apoptosis and kinase activation is unknown. Studies were done in rat hepatocytes, HUH7 cells, and HUH7 cells stably transfected with rat Ntcp (HUH7-Ntcp). Cells were treated with GCDC and apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for the active cleaved fragment of caspase 3. Kinase activation was determined by immunoblotting with phospho-specific antibodies. JTE-013, an inhibitor of S1PR2, significantly attenuated morphological evidence of GCDC-induced apoptosis and prevented caspase 3 cleavage in rat hepatocytes and HUH7-Ntcp cells. In hepatocytes, JTE-013 mildly suppressed, augmented, and had no effect on GCDC-induced JNK, Akt, and Erk phosphorylation, respectively. Similar results were seen in HUH7-Ntcp cells except for mild suppression of JNK and Erk phosphorylation. Knockdown of S1PR2 in HUH7-Ntcp augmented Akt, inhibited JNK, and had no effect on Erk phosphorylation. GCDC failed to induce apoptosis or kinase activation in HUH7 cells. In conclusion, SIPR2 inhibition attenuates GCDC-induced apoptosis and inhibits and augments GCDC-induced JNK and Akt phosphorylation, respectively. In addition, GCDC must enter hepatocytes to mediate cell death or activate kinases. These results suggest that SIPR2 activation is proapoptotic in GCDC-induced cell death but that this effect is not due to direct ligation of the S1PR2 by the bile acid.

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In hepatocytes, cAMP both activates p38 mitogen-activated protein kinase (MAPK) and increases the amount of multidrug resistance-associated protein-2 (MRP2) in the plasma membrane (PM-MRP2). Paradoxically, taurolithocholate (TLC) activates p38 MAPK but decreases PM-MRP2 in hepatocytes. These opposing effects of cAMP and TLC could be mediated via different p38 MAPK isoforms (α and ß) that are activated differentially by upstream kinases (MKK3, MKK4, and MKK6). Thus we tested the hypothesis that p38α MAPK and p38ß MAPK mediate increases and decreases in PM-MRP2 by cAMP and TLC, respectively. Studies were conducted in hepatocytes isolated from C57BL/6 wild-type (WT) and MKK3-knockout (MKK3(-/-)) mice and in a hepatoma cell line (HuH7) that overexpresses sodium-taurocholate cotransporting polypeptide (NTCP) (HuH-NTCP). Cyclic AMP activated MKK3, p38 MAPK, and p38α MAPK and increased PM-MRP2 in WT hepatocytes, but failed to activate p38α MAPK or increase PM-MRP2 in MKK3(-/-) hepatocytes. In contrast to cAMP, TLC activated total p38 MAPK but decreased PM-MRP2, and did not activate MKK3 or p38α MAPK in WT hepatocytes. In MKK3(-/-) hepatocytes, TLC still decreased PM-MRP2 and activated p38 MAPK, indicating that these effects are not MKK3-dependent. Additionally, TLC activated MKK6 in MKK3(-/-) hepatocytes, and small interfering RNA knockdown of p38ß MAPK abrogated TLC-mediated decreases in PM-MRP2 in HuH-NTCP cells. Taken together, these results suggest that p38α MAPK facilitates plasma membrane insertion of MRP2 by cAMP, whereas p38ß MAPK mediates retrieval of PM-MRP2 by TLC.

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Retained bile acids, which are capable of inducing cell death, activate protein kinase Cδ (PKC-δ) in hepatocytes. In nonhepatic cells, both pro- and antiapoptotic effects of PKC-δ are described. The aim of this study was to determine the role of PKC-δ in glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes and human HUH7-Na-taurocholate-cotransporting polypeptide (Ntcp) cells. Apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for caspase 3 cleavage. The role of PKC-δ was evaluated with a PKC activator (phorbol myristate acetate, PMA) and PKC inhibitors (chelerythrine, H-7, or calphostin), PKC-δ knockdown, and wild-type (WT) or constitutively active (CA) PKC-δ. PKC-δ activation was monitored by immunoblotting for PKC-δ Thr505 and Tyr311 phosphorylation or by membrane translocation. JNK and Akt phosphorylation and the amount of total bisindolylmaleimide (BIM) were determined by immunoblotting. GCDC induced the translocation of PKC-δ to the mitochondria and/or plasma membrane in rat hepatocytes and HUH7-Ntcp cells and increased PKC-δ phosphorylation on Thr505, but not on Tyr311, in HUH7-Ntcp cells. GCDC-induced apoptosis was attenuated by PMA and augmented by PKC inhibition in rat hepatocytes. In HUH-Ntcp cells, transfection with CA or WT PKC-δ attenuated GCDC-induced apoptosis, whereas knockdown of PKC-δ increased GCDC-induced apoptosis. PKC-δ silencing increased GCDC-induced JNK phosphorylation, decreased GCDC-induced Akt phosphorylation, and increased expression of BIM. GCDC translocated BIM to the mitochondria in rat hepatocytes, and knockdown of BIM in HUH7-Ntcp cells decreased GCDC-induced apoptosis. Collectively, these results suggest that PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, Akt activation, and downregulation of BIM.

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Rab proteins (Ras homologous for brain) play an important role in vesicle trafficking. Rab4 and Rab11 are involved in vesicular trafficking to the plasma membrane from early endosomes and recycling endosomes, respectively. Tauroursodeoxycholate (TUDC) and cAMP increase bile formation, in part, by increasing plasma membrane localization of multidrug resistance-associated protein 2 (MRP2). The goal of the present study was to determine the role of these Rab proteins in the trafficking of MRP2 by testing the hypothesis that Rab11 and/or Rab4 facilitate cAMP- and TUDC-induced MRP2 translocation to the plasma membrane. Studies were conducted in HuH-NTCP cells (HuH7 cells stably transfected with human NTCP), which constitutively express MRP2. HuH-NTCP cells were transfected with Rab11-WT and GDP-locked dominant inactive Rab11-GDP or with Rab4-GDP to study the role of Rab11 and Rab4. A biotinylation method and a GTP overlay assay were used to determine plasma membrane MRP2 and activation of Rab proteins (Rab11 and Rab4), respectively. Cyclic AMP and TUDC increased plasma membrane MRP2 and stimulated Rab11 activity. Plasma membrane translocation of MRP2 by cAMP and TUDC was increased and inhibited in cells transfected with Rab11-WT and Rab11-GDP, respectively. Cyclic AMP (previous study) and TUDC increased Rab4 activity. However, cAMP- and TUDC-induced increases in MRP2 were not inhibited by Rab4-GDP. Taken together, these results suggest that Rab11 is involved in cAMP- and TUDC-induced MRP2 translocation to the plasma membrane.

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Transhepatic solute transport provides the osmotic driving force for canalicular bile formation. Choleretic and cholestatic agents affect bile formation, in part, by altering plasma membrane localizations of transporters involved in bile formation. These short-term dynamic changes in transporter location are highly regulated posttranslational events requiring various cellular signaling pathways. Interestingly, both choleretic and cholestatic agents activate the same intracellular signaling kinases, such as phosphoinositide-3-kinase (PI3K), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK). An emerging theme is that choleretic and cholestatic effects may be mediated by different isoforms of these kinases. This is most evident for PKC-mediated regulation of plasma membrane localization of Na+-taurocholate cotransporting polypeptide (NTCP) and multidrug resistance-associated protein 2 (MRP2) by conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ). aPKCζ may mediate choleretic effects by inserting NTCP into the plasma membrane, and nPKCε may mediate cholestatic effects by retrieving MRP2 from the plasma membrane. On the other hand, cPKCα and nPKCδ may be involved in choleretic, cholestatic, and anticholestatic effects by inserting, retrieving, and inhibiting retrieval of transporters, respectively. The effects of PKC isoforms may be mediated by phosphorylation of the transporters, actin binding proteins (radixin and myristoylated alanine-rich C kinase substrate), and Rab proteins. Human NTCP plays an important role in the entry of hepatitis B and D viruses into hepatocytes and consequent infection. Thus, PKCs, by regulating NTCP trafficking, may also play an important role in hepatic viral infections.

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The SLC10A transporter gene family consists of seven members and substrates transported by three members (SLC10A1, SLC10A2 and SLC10A6) are Na(+)-dependent. SLC10A1 (sodium taurocholate cotransporting polypeptide [NTCP]) and SLC10A2 (apical sodium-dependent bile salt transporter [ASBT]) transport bile salts and play an important role in maintaining enterohepatic circulation of bile salts. Solutes other than bile salts are also transported by NTCP. However, ASBT has not been shown to be a transporter for non-bile salt substrates. While the transport function of NTCP can potentially be used as liver function test, interpretation of such a test may be complicated by altered expression of NTCP in diseases and presence of drugs that may inhibit NTCP function. Transport of bile salts by NTCP and ASBT is inhibited by a number of drugs and it appears that ASBT is more permissive to drug inhibition than NTCP. The clinical significance of this inhibition in drug disposition and drug-drug interaction remains to be determined. Both NCTP and ASBT undergo post-translational regulations that involve phosphorylation/dephosphorylation, translocation to and retrieval from the plasma membrane and degradation by the ubiquitin-proteasome system. These posttranslational regulations are mediated via signaling pathways involving cAMP, calcium, nitric oxide, phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) and protein phosphatases. There appears to be species difference in the substrate specificity and the regulation of plasma membrane localization of human and rodent NTCP. These differences should be taken into account when extrapolating rodent data for human clinical relevance and developing novel therapies. NTCP has recently been shown to play an important role in HBV and HDV infection by serving as a receptor for entry of these viruses into hepatocytes.

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The Na(+) taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.

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OBJECTIVE: To investigate whether differences existed between clinically normal dogs and dogs with goniodysgenesis-related glaucoma (GDRG) in serum autoantibodies against optic nerve antigens. ANIMALS: 16 dogs with GDRG, 17 healthy dogs with unremarkable pectinate ligament and iridocorneal angle morphology, and 13 euthanized dogs with no major ocular abnormalities or underlying diseases. PROCEDURES: Western blotting was performed with optic nerve extracts from the euthanized dogs as an antigen source and serum from clinically normal dogs and dogs with GDRG as a primary antibody (autoantibody) source. Blots were evaluated for presence and density of bands. RESULTS: Multiple bands were identified on western blots from all dogs with GDRG and all clinically normal dogs, with a high degree of variability among individual dogs. Dogs with GDRG were significantly more likely than healthy dogs to have bands present at 38, 40, and 68 kDa. Dogs with GDRG had significant increases in autoreactivity at 40 and 53 kDa and a significant decrease in autoreactivity at 48 kDa. CONCLUSIONS AND CLINICAL RELEVANCE: Significant differences in serum autoantibodies against optic nerve antigens were found in dogs with versus without GDRG. Although it remains unclear whether these differences were part of the pathogenesis of disease or were sequelae to glaucomatous changes, these findings provide support for the hypothesis that immune-mediated mechanisms play a role in the development or progression of GDRG. However, the high degree of variability among individual dogs and the considerable overlap between groups suggest that the clinical usefulness of this technique for distinguishing dogs with GDRG from clinically normal dogs is likely limited.

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UNLABELLED: Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug-resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine-rich C kinase substrate (MARCKS) is a membrane-bound F-actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine-rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCϵ-mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in rat hepatocytes. TLC increased PM-PKCϵ and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. cAMP did not affect PM-PKCϵ and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant-negative (DN) PKCϵ reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCϵ, and this suggested PKCϵ-mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation-deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM-MRP2. CONCLUSION: Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC-mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane.

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Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.

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The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na(+)-TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCP-dependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na(+)-dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V(max) but not the K(m) of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP.

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BACKGROUND & AIMS: Glycochenodeoxycholate (GCDC) and taurolithocholate (TLC) are hepatotoxic and cholestatic bile salts, whereas tauroursodeoxycholate (TUDC) is cytoprotective and anticholestatic. Yet they all act, in part, through phosphatidylinositol-3-kinase(PI3K)-dependent mechanisms ("PI3K-paradox"). Hepatocytes express three catalytic PI3K Class I isoforms (p110α/ß/γ), specific functions of which, in liver, are unclear. In other cell types, p110γ is associated with detrimental effects. To shed light on the PI3K enigma, we determined whether hydrophobic and hydrophilic bile salts differentially activate distinct p110 isoforms in hepatocytes, and whether p110γ mediates bile salt-induced hepatocyte cell death. METHODS: Isoform-specific PI3K activity assays were established to determine isoform activation by bile salts in rat hepatocytes. Activation of Akt and JNK was determined by immunoblotting. Following stimulation with hydrophobic bile salts, hepatocellular apoptosis was determined morphologically after Hoechst staining and by analysis of caspase-3/-7 activity or caspase-3 cleavage. Activity or expression of PI3K p110γ was inhibited pharmacologically (AS604850) or by knock-down using specific siRNA. RESULTS: All bile salts tested activated p110ß, while p110α was activated by TUDC and GCDC. Intriguingly, only hydrophobic bile salts activated p110γ. Inhibition of p110γ attenuated GCDC-induced Akt- and JNK-activation, but did not alter TUDC- or cAMP-induced Akt-signaling in rat hepatocytes. Inhibition or knock-down of p110γ markedly attenuated hydrophobic bile salt-induced apoptosis in rat hepatocytes and human hepatoma cell lines but did not alter Fas-, tumor necrosis factor α- and etoposide-induced apoptosis. Depletion of Ca(++) prevented GCDC-induced toxicity in rat hepatocytes but did not affect GCDC-induced Akt- and JNK-activation. CONCLUSIONS: PI3K p110γ is activated by hydrophobic, but not hydrophilic bile salts. Bile salt-induced hepatocyte apoptosis is partly mediated via a PI3K p110γ dependent signaling pathway, potentially involving JNK.

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