Hepatic Ca2 Research Articles

NCLX controls hepatic mitochondrial Ca2+ extrusion and couples hormone-mediated mitochondrial Ca2+ oscillations with gluconeogenesis

ObjectiveHepatic Ca2+ signaling has been identified as a crucial key factor in driving gluconeogenesis. The involvement of mitochondria in hormone-induced Ca2+ signaling and their contribution to metabolic activity remain, however, poorly understood. Moreover, the molecular mechanism governing the mitochondrial Ca2+ efflux signaling remains unresolved. This study investigates the role of the Na+/Ca2+ exchanger, NCLX, in modulating hepatic mitochondrial Ca2+ efflux, and examines its physiological significance in hormonal hepatic Ca2+ signaling, gluconeogenesis, and mitochondrial bioenergetics. MethodsPrimary mouse hepatocytes from both an AAV-mediated conditional hepatic-specific and a total mitochondrial Na+/Ca2+ exchanger, NCLX, knockout (KO) mouse models were employed for fluorescent monitoring of purinergic and glucagon/vasopressin-dependent mitochondrial and cytosolic hepatic Ca2+ responses in cultured hepatocytes. Isolated liver mitochondria and permeabilized primary hepatocytes were used to analyze the ion-dependence of Ca2+ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed by first monitoring glucose levels in fasted mice, and subsequently subjecting the mice to a pyruvate tolerance test while monitoring their blood glucose. Additionally, cultured primary hepatocytes from both genotypes were assessed in vitro for glucagon-dependent glucose production and cellular bioenergetics through glucose oxidase assay and Seahorse respirometry, respectively. ResultsAnalysis of Ca2+ responses in isolated liver mitochondria and cultured primary hepatocytes from NCLX KO versus WT mice showed that NCLX serves as the principal mechanism for mitochondrial calcium extrusion in hepatocytes. We then determined the role of NCLX in glucagon and vasopressin-induced Ca2+ oscillations. Consistent with previous studies, glucagon and vasopressin triggered Ca2+ oscillations in WT hepatocytes, however, the deletion of NCLX resulted in selective elimination of mitochondrial, but not cytosolic, Ca2+ oscillations, underscoring NCLX’s pivotal role in mitochondrial Ca2+ regulation. Subsequent in vivo investigation for hepatic NCLX role in gluconeogenesis revealed that, as opposed to WT mice which maintained normoglycemic blood glucose levels when fasted, conditional hepatic-specific NCLX KO mice exhibited a faster drop in glucose levels, becoming hypoglycemic. Furthermore, KO mice showed deficient conversion of pyruvate to glucose when challenged under fasting conditions. Concurrent in vitro assessments showed impaired glucagon-dependent glucose production and compromised bioenergetics in KO hepatocytes, thereby underscoring NCLX’s significant contribution to hepatic glucose metabolism. ConclusionsThe study findings demonstrate that NCLX acts as the primary Ca2+ efflux mechanism in hepatocytes. NCLX is indispensable for regulating hormone-induced mitochondrial Ca2+ oscillations, mitochondrial metabolism, and sustenance of hepatic gluconeogenesis.

When gluttony strikes: how high-fat diet impairs hepatic Ca2+ signalling.

When gluttony strikes: how high-fat diet impairs hepatic Ca2+ signalling.

Short-term high-fat diet feeding of mice suppresses catecholamine-stimulated Ca2+ signalling in hepatocytes and intact liver.

Excess consumption of carbohydrates, fat and calories leads to non-alcoholic fatty liver disease (NAFLD) and hepatic insulin resistance; these are major factors in the pathogenesis of type II diabetes. Hormones and catecholamines acting through G-protein coupled receptors (GPCRs) linked to phospholipase C (PLC) and increases in cytosolic Ca2+ ([Ca2+ ]c ) regulate many metabolic functions of the liver. In the intact liver, catabolic hormones such as glucagon, catecholamines and vasopressin integrate and synergize to regulate the frequency and extent to which [Ca2+ ]c waves propagate across hepatic lobules to control metabolism. Dysregulation of hepatic Ca2+ homeostasis has been implicated in the development of metabolic disease, but changes in hepatic GPCR-dependent Ca2+ signalling have been largely unexplored in this context. We show that short-term, 1-week, high-fat diet (HFD) feeding of mice attenuates noradrenaline-stimulated Ca2+ signalling, reducing the number of cells responding and suppressing the frequency of [Ca2+ ]c oscillations in both isolated hepatocytes and intact liver. The 1-week HFD feeding paradigm did not change basal Ca2+ homeostasis; endoplasmic reticulum Ca2+ load, store-operated Ca2+ entry and plasma membrane Ca2+ pump activity were unchanged compared to low-fat diet (LFD)-fed controls. However, noradrenaline-induced inositol 1,4,5-trisphosphate production was significantly reduced after HFD feeding, demonstrating an effect of HFD on receptor-stimulated PLC activity. Thus, we have identified a lesion in the PLC signalling pathway induced by short-term HFD feeding, which interferes with hormonal Ca2+ signalling in isolated hepatocytes and the intact liver. These early events may drive adaptive changes in signalling, which lead to pathological consequences in fatty liver disease. KEY POINTS: Non-alcoholic fatty liver disease (NAFLD) is a growing epidemic. In healthy liver, the counteracting effects of catabolic and anabolic hormones regulate metabolism and energy storage as fat. Hormones and catecholamines promote catabolic metabolism via increases in cytosolic Ca2+ ([Ca2+ ]c ). We show that 1week high-fat diet (HFD) feeding of mice attenuated the Ca2+ signals induced by physiological concentrations of noradrenaline. Specifically, HFD suppressed the normal pattern of periodic [Ca2+ ]c oscillations in isolated hepatocytes and disrupted the propagation of intralobular [Ca2+ ]c waves in the intact perfused liver. Short-term HFD inhibited noradrenaline-induced inositol 1,4,5-trisphosphate generation, but did not change basal endoplasmic reticulum Ca2+ load or plasma membrane Ca2+ fluxes. We propose that impaired Ca2+ signalling plays a key role in the earliest phases of the etiology of NAFLD, and is responsible for many of the ensuing metabolic and related dysfunctional outcomes at the cellular and whole tissue level.

English

Lead and di-(2-ethylhexyl) phthalate (DEHP) are common environmental toxicants of concern around the world. Although effects of individual exposures to both agents are well documented, there is a dearth of information on the effects of co-exposure to both agents. In this study, combined exposure to lead and DEHP was investigated for effects on ATPase activities in the liver, brain and kidney tissues of rats. Male albino rats were daily exposed to either 200 ppm lead as lead acetate in their drinking water and/or 100 mg DEHP kg-1 body weight in olive oil by gastric intubation for 30 days. Changes in total body weight, relative organ weights as well as brain, hepatic and renal activities of total, Na+K+ -, Ca2+ - and Mg2+-ATPases were used as biomarkers of toxicity. Hepatomegaly and brain atrophy heralded exposure to both agents. Individual exposure to lead and DEHP resulted in reduction in hepatic Ca2+- and Mg2+- ATPase activities but no significant effect on total ATPase activity, however combined exposure produced significant activation of Ca2+-, Na+K+- and total ATPase while restoring Mg2+ - ATPase towards control. A potentiating effect on lead by DEHP was observed in hepatic Na+K+ - ATPase. Lead stimulated the activities of renal Ca2+- and total ATPases while DEHP on the contrary caused significant reduction in total ATPase activity and no significant effects on Ca2+- ATPase activity. Co-treatment produced antagonistic effects leading to normal renal Ca2+- and total ATPase activities. Brain Na+K+ -, Ca2+ - and total ATPase activities were depressed in co-exposure while Mg2+ - ATPase was up-regulated. Lead potentiated DEHP-induced inhibition of brain total - ATPase while co-treatment produced antagonistic effects on brain Ca2+ - ATPase. The findings of this study highlight organ specific variations in response to combined lead and DEHP exposure in rats. Key words: Hepatotoxic, neurotoxic, ATPases, DEHP, lead, co-exposure, hepatomegaly.

ASBT Vivo‐Morpholino Decreases Hepatic Mast Cell, Fibrosis and Biliary Senescence in Mdr2 ‐/‐ Mice

Background Primary Sclerosing Cholangitis (PSC) is characterized by increased biliary senescence, liver fibrosis, and mast cell (MC) infiltration. Mdr2-/- mice, model of PSC, mimic some features of PSC. PSC features include (i) increased conjugated total bile acid (TBA) levels, (ii) dysregulation of BA synthesis and (iii) higher hepatic expression of the apical sodium bile acid transporter (ASBT), expressed only by cholangiocytes. Hydrophobic BAs enhance cholestasis, biliary proliferation and ASBT expression. MCs infiltrate the liver in PSC and reside near senescent cholangiocytes, increasing biliary damage and TBA levels. ASBT is regulated by farnesoid X receptor (FXR) and blocking MC-FXR decreases biliary damage, fibrosis, and biliary ASBT expression. No information exists to demonstrate if blocking hepatic ASBT inhibits MC activation and subsequent liver damage in Mdr2-/- mice. Methods 12 wk male Mdr2−/− mice were treated with control or ASBT Vivo-Morpholino (2 injections, 12.5 mg oligo/kg BW via tail vein for 1 wk). Liver damage was determined by H&E and serum enzyme levels. Ductular reaction (DR) was evaluated by immunohistochemistry (IHC) for CK-19, Ki-67 and immunofluorescence (IF) for p16, co-stained with CK-19. ASBT expression was measured by qPCR and IF in liver, isolated cholangiocytes and small intestine. MC infiltration was measured by IHC for Tryptase β-2 in liver and intestine. Hepatic fibrosis was determined by Fast Green-Sirius Red staining. TBA levels were measured in serum, liver and feces. BA receptors/transporters were co-stained with CK-19 in small intestine and liver. Results Mdr2−/− mice have increased DR, fibrosis, and serum/hepatic TBA levels, which decrease in Mdr2−/− ASBT Vivo-Morpholino mice. Inhibition of ASBT in Mdr2-/- mice reduced (i) biliary and intestinal ASBT expression and (ii) serum and hepatic CA and CDCA levels. Fecal TBA levels decreased, but CA and CDCA levels increased, following ASBT Vivo-Morpholino. ASBT Vivo-Morpholino treatment reduced hepatic and intestinal MC-infiltration/activation. Conclusion Elevated serum TBA levels seen in PSC lead to increased intestinal BA transport inducing MC infiltration and increased biliary ASBT. Inhibition of biliary ASBT blocks hepatic and intestinal MC recruitment and subsequent DR, hepatic fibrosis and TBA levels. Targeting MCs via ASBT inhibition is a novel strategy for the management of PSC.

A Villin-Driven Fxr Transgene Modulates Enterohepatic Bile Acid Homeostasis and Response to an n-6-Enriched High-Fat Diet.

A diet high in n-6 polyunsaturated fatty acids (PUFAs) may contribute to inflammation and tissue damage associated with obesity and pathologies of the colon and liver. One contributing factor may be dysregulation by n-6 fatty acids of enterohepatic bile acid (BA) metabolism. The farnesoid X receptor (FXR) is a nuclear receptor that regulates BA homeostasis in the liver and intestine. This study aims to compare the effects on FXR regulation and BA metabolism of a palm oil-based diet providing 28% energy (28%E) from fat and low n-6 linoleic acid (LA, 2.5%E) (CNTL) with those of a soybean oil-based diet providing 50%E from fat and high (28%E) in LA (n-6HFD). Wild-type (WT) littermates and a transgenic mouse line overexpressing the Fxrα1 isoform under the control of the intestine-specific Villin promoter (Fxrα1TG) were fed the CNTL or n-6HFD starting at weaning through 16 weeks of age. Compared to the CNTL diet, the n-6HFD supports higher weight gain in both WT and FxrαTG littermates; increases the expression of Fxrα1/2, and peroxisome proliferator-activated receptor-γ1 (Pparγ1) in the small intestine, Fxrα1/2 in the colon, and cytochrome P4507A1 (Cyp7a1) and small heterodimer protein (Shp) in the liver; and augments the levels of total BA in the liver, and primary chenodeoxycholic (CDCA), cholic (CA), and β-muricholic (βMCA) acid in the cecum. Intestinal overexpression of the Fxra1TG augments expression of Shp and ileal bile acid-binding protein (Ibabp) in the small intestine and Ibabp in the proximal colon. Conversely, it antagonizes n-6HFD-dependent accumulation of intestinal and hepatic CDCA and CA; hepatic levels of Cyp7a1; and expression of Pparγ in the small intestine. We conclude that intestinal Fxrα1 overexpression represses hepatic de novo BA synthesis and protects against n-6HFD-induced accumulation of human-specific primary bile acids in the cecum.

Uncovering the links between osteoporosis and diabetes

Introduction: Using an animal model for osteopenia to describe the distribution of Lead (Pb) throughout the body, we have reported that ovariectomized animals had significantly lower serum osteocalcin (OC) levels and higher, although nonsignificant, serum C-terminal peptide-bound crosslinks of collagen (CTX) levels than intact animals [1]. Here we present unpublished data on concentrations of elements of interest (Ca, Cl, Fe, K, Na and Pb) in hard and soft tissues of the same animals, to uncover the links between osteoporosis and diabetes. The model makes it feasible to investigate such links, therefore the appropriateness of this approach is not only warranted but also justified by the 3RS principle for the use of animals in research. Materials and methods: The animal study was approved by the Ethics Committee of ISCSEM and was conducted following the Portuguese Law and EU Directive 2010/63/EU, in a licensed animal facility. Two groups of adult female Wistar rats of the same age were considered: normal intact rats (n = 7); and OVX animals (n = 7) ovariectomized between the 11th and 12th week of age. From age 6-months on, all animals received the same dose of Pb acetate in water (500 ppm) for 6 months. Tissues collected for analysis included lumbar vertebrae and femora bones, as well as blood and liver. The concentration of elements of interest in hard and soft tissues was determined by Wavelength Dispersive X Ray Fluorescence (WDXRF) spectroscopy, using a 4kW commercial spectrometer (S4 Pioneer, Bruker AXS). Multivariate analysis of variance (MANOVA) was used in group comparisons, given the multiple correlations between elemental concentrations. Results: MANOVA methods show that OVX animals have significantly higher liver Cl, Na and Ca concentrations, whereas normal animals show significantly higher liver Pb and K concentrations, with observed power of 91.0%. Significantly lower serum OC levels were observed in the OVX group, with observed power of 72.7%, with no differences between groups for CTX levels. Lower Ca content was strongly suggested for trabecular bone of OVX animals. Discussion and conclusions: Significant differences in hepatic Ca concentration between OVX and normal animals suggest that Ca signalling mechanisms regulating gluconeogenesis, through modulation of hormones such as glucagon, insulin, vasopressin and adrenalin [2,3], may occur differently in the two groups. Moreover, since extracellular concentrations of cations such as K and Na markedly affect the hormonal activity effects on gluconeogenesis [4], the observed differences in the hepatic concentrations of such elements between the groups may further increase differences in gluconeogenesis in the two groups. Therefore, we postulated that bone turnover changes may impact on gluconeogenesis, which launched the basis for another research project linking osteoporosis to diabetes, nearly concluded in our laboratory.

Defective STIM-mediated store operated Ca2+ entry in hepatocytes leads to metabolic dysfunction in obesity.

Defective Ca2+ handling is a key mechanism underlying hepatic endoplasmic reticulum (ER) dysfunction in obesity. ER Ca2+ level is in part monitored by the store-operated Ca2+ entry (SOCE) system, an adaptive mechanism that senses ER luminal Ca2+ concentrations through the STIM proteins and facilitates import of the ion from the extracellular space. Here, we show that hepatocytes from obese mice displayed significantly diminished SOCE as a result of impaired STIM1 translocation, which was associated with aberrant STIM1 O-GlycNAcylation. Primary hepatocytes deficient in STIM1 exhibited elevated cellular stress as well as impaired insulin action, increased glucose production and lipid droplet accumulation. Additionally, mice with acute liver deletion of STIM1 displayed systemic glucose intolerance. Conversely, over-expression of STIM1 in obese mice led to increased SOCE, which was sufficient to improve systemic glucose tolerance. These findings demonstrate that SOCE is an important mechanism for healthy hepatic Ca2+ balance and systemic metabolic control.

Kampo formula "Hochu-ekki-to" suppressed carbon tetrachloride-induced hepatotoxicity in mice.

The aim of this study was to investigate whether pretreatment with the Japanese herbal medicine "Hochu-ekki-to" (TJ-41) has an ameliorative effect on carbon tetrachloride (CCl4)-induced hepatotoxicity through anorexia prevention. Twenty-four hours before CCl4 injection, TJ-41 or saline solution was intraperitoneally administered. Furthermore, 24h after TJ-41 injection, mice were intraperitoneally administered 1.6g/kg CCl4 or olive oil. Moreover, 24h after CCl4/olive oil injection, mice from each group were euthanized and bled for plasma analysis. Mice injected with CCl4 exhibited severe anorexia. Moreover, CCl4 increased the plasma levels of hepatic injury markers (i.e., alanine aminotransferase and aspartate aminotransferase) as well as lipid peroxidation and hepatic Ca levels. Pretreatment with TJ-41 recovered the CCl4-induced anorexia and plasma levels of the hepatic injury markers. Moreover, CCl4-induced lipid peroxidation and hepatic Ca levels decreased upon TJ-41 pretreatment. In addition, hepatic metallothionein levels in the TJ-41+CCl4-treated group were decreased by >50% compared with the levels in the TJ-41-treated group, implying that metallothionein was consumed by CCl4-induced radicals. Our results suggest that TJ-41 attenuates CCl4-induced hepatotoxicity, presumably by the induction of metallothionein, which in turn scavenges radicals induced by CCl4 exposure.

Suppressive Effect of Kampo Formula “Juzen-taiho-to” on Carbon Tetrachloride-Induced Hepatotoxicity in Mice

The aim of the present study was to investigate whether pretreatment with the Japanese herbal medicine, "Juzen-taiho-to" (JTX), had an ameliorative effect on carbon tetrachloride (CCl4)-induced hepatotoxicity through anorexia prevention. Mice injected with CCl4 exhibited severe anorexia. Moreover, CCl4 increased the plasma levels of hepatic injury markers (i.e., alanine aminotransferase and aspartate aminotransferase), lipid peroxidation, and hepatic Ca(2+) levels. Pretreatment with JTX recovered the CCl4-induced anorexia. In addition, JTX pretreatment decreased CCl4-induced plasma levels of hepatic injury markers. Increased Ca(2+) is a known indicator of the final progression to hepatocyte death, and CCl4-induced hepatotoxicity is mainly caused by oxidative stress. The present study indicated CCl4-induced lipid peroxidation and hepatic Ca(2+) content decreased with JTX pretreatment. Our results suggest that JTX has potential to protect of CCl4-induced anorexia, and the modulation of oxidative stress.

Baicalin Attenuates High Fat Diet-Induced Obesity and Liver Dysfunction: Dose-Response and Potential Role of CaMKKβ/AMPK/ACC Pathway

Background/Aims: Obesity-associated fatty liver disease affects millions of individuals. This study aimed to evaluate the therapeutic effects of baicalin to treat obesity and fatty liver in high fat diet-induced obese mice, and to study the potential molecular mechanisms. Methods: High fat diet-induced obese animals were treated with different doses of baicalin (100, 200 and 400 mg/kg/d). Whole body, fat pad and liver were weighed. Hyperlipidemia, liver steatosis, liver function, and hepatic Ca²⁺/CaM-dependent protein kinase kinase β (CaMKKβ) / AMP-activated protein kinase (AMPK) / acetyl-CoA carboxylase (ACC) were further evaluated. Results: Baicalin significantly decreased liver, epididymal fat and body weights in high fat diet-fed mice, which were associated with decreased serum levels of triglycerides, total cholesterol, LDL, alanine transaminase and aspartate transaminase, but increased serum HDL level. Pathological analysis revealed baicalin dose-dependently decreased the degree of hepatic steatosis, with predominantly diminished macrovesicular steatosis at lower dose but both macrovesicular and microvesicular steatoses at higher dose of baicalin. Baicalin dose-dependently inhibited hepatic CaMKKβ/AMPK/ACC pathway. Conclusion: These data suggest that baicalin up to 400 mg/kg/d is safe and able to decrease the degree of obesity and fatty liver diseases. Hepatic CaMKKβ/AMPK/ACC pathway may mediate the therapeutic effects of baicalin in high fat diet animal model.

Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes

In hepatocytes, as in other cell types, Ca 2+ signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP 3R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca 2+ signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP 3 receptors (InsP 3R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP 3R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP 3-induced Ca 2+ release in hepatocytes. This can be explained by the rather low expression level expression of InsP 3R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca 2+ signaling via an inhibitory effect on InsP 3 synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP 3 synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.

Copper inhibits P2Y 2-dependent Ca 2+ signaling through the effects on thapsigargin-sensitive Ca 2+ stores in HTC hepatoma cells

Purinergic P2Y 2 G-protein coupled receptors play a key role in the regulation of hepatic Ca 2+ signaling by extracellular ATP. The concentration of copper in serum is about 20 μM. Since copper accumulates in the liver in certain disease states, the purpose of these studies was to assess the effects of copper on P2Y 2 receptors in a model liver cell line. Exposure to a P2Y 2 agonist UTP increased [Ca 2+] i by stimulating Ca 2+ release from thapsigargin-sensitive Ca 2+ stores. Pretreatment of HTC cells for several minutes with copper did not affect cell viability, but potently inhibited increases in [Ca 2+] i evoked by UTP and thapsigargin. During this pretreatment, copper was not transported into the cytosol, and inhibited P2Y 2 receptors in a concentration-dependent manner with the IC 50 of about 15 μM. These results suggest that copper inhibits P2Y 2 receptors through the effects on thapsigargin-sensitive Ca 2+ stores by acting from an extracellular side. Further experiments indicated that these effect of copper may lead to inhibition of regulatory volume decrease (RVD) evoked by hypotonic solution. Thus, copper may contribute to defective regulation of purinergic signaling and liver cell volume in diseases associated with the increased serum copper concentration.

In vivo treatment with stobadine prevents lipid peroxidation, protein glycation and calcium overload but does not ameliorate Ca 2+-ATPase activity in heart and liver of streptozotocin-diabetic rats: comparison with vitamin E

Hyperglycemia leads to excess production of reactive oxygen species (ROS), lipid peroxidation and protein glycation that may impair cellular calcium homeostasis and results in calcium sequestration and dysfunction in diabetic tissues. Stobadine (ST) is a pyridoindole antioxidant has been postulated as a new cardio- and neuroprotectant. This study was undertaken to test the hypothesis that the treatment with ST inhibits calcium accumulation, reduces lipid peroxidation and protein glycation and can change Ca 2+,Mg 2+-ATPase activity in diabetic animals. The effects of vitamin E treatment were also evaluated and compared with the effects of combined treatment with ST. Diabetes was induced by streptozotocin (STZ, 55 mg/kg i.p.). Some of diabetic rats and their age-matched controls were treated orally with a low dose of ST (24.7 mg/kg/day), vitamin E (400–500 IU/kg/day) or ST plus vitamin E for 10 weeks. ST and vitamin E separately produced, in a similar degree, reduction in diabetes-induced hyperglycemia. Each antioxidant alone significantly lowered the levels of plasma lipid peroxidation, cardiac and hepatic protein glycation in diabetic rats but vitamin E treatment was found to be more effective than ST treatment alone. Diabetes-induced increase in plasma triacylglycerol levels was not significantly altered by vitamin E treatment but markedly reduced by ST alone. The treatment with each antioxidant completely prevented calcium accumulation in diabetic heart and liver. Microsomal Ca 2+,Mg 2+-ATPase activity significantly decreased in both tissues of untreated diabetic rats. ST alone significantly increased microsomal Ca 2+,Mg 2+-ATPase activity in the heart of normal rats. However, neither treatment with ST nor vitamin E alone, nor their combination did change cardiac Ca 2+,Mg 2+-ATPase activity in diabetic heart. In normal rats, neither antioxidant had a significant effect on hepatic Ca 2+,Mg 2+-ATPase activity. Hepatic Ca 2+,Mg 2+-ATPase activity of diabetic rats was not changed by single treatment with ST, while vitamin E alone completely prevented diabetes-induced inhibition in microsomal Ca 2+,Mg 2+-ATPase activity in liver. Combined treatment with ST and vitamin E provided more benefits in the reduction of hyperglycemia and lipid peroxidation in diabetic animals. This study describes potential mechanisms on cellular effects of ST in the presence of diabetes-induced hyperglycemia that may delay or inhibit the development of diabetic complications. The use of ST together with vitamin E can better control hyperglycemia-induced oxidative stress.

Interspecies differences in hepatic Ca(2+)-ATPase activity and the effect of cold preservation on porcine liver Ca(2+)-ATPase function.

The accumulation of intracellular calcium ([Ca(2+)](i)) caused by ischemia-reperfusion during liver transplantation has been implicated as a factor leading to primary graft nonfunction. Plasma membrane (PM) and endoplasmic reticulum (ER) Ca(2+)-adenosinetriphosphatases (ATPases) are the primary transporters that maintain [Ca(2+)](i) homeostasis in the liver. We hypothesized that the porcine liver is better than the rat liver as a model for the study of human liver Ca(2+)-ATPase activity. We also hypothesized that cold preservation would depress Ca(2+)-ATPase activity in the porcine liver. Pig and rat livers were harvested, and human liver samples were obtained from surgical resection specimens. All were preserved with University of Wisconsin solution, and porcine livers were also preserved on ice for 2 to 18 hours. Ca(2+)-ATPase activity was measured after incubation with (45)Ca(2+) and adenosine triphosphate in the presence of specific Ca(2+)-ATPase inhibitors. Porcine PM and ER Ca(2+)-ATPase activities were 0.47 +/- 0.03 and 1.57 +/- 0.10 nmol of Ca(2+)/mg of protein/min, respectively. This was not significantly different from human liver, whereas rat liver was significantly greater at 2.60 +/- 0.03 and 9.2 +/- 0.9 nmol of Ca(2+)/mg of protein/min, respectively. We conclude that the Ca(2+)-ATPase activity in the pig liver is equivalent to that of human liver, and thus, the pig liver is a better model than the rat liver. Cold preservation studies showed a significant decrease in porcine hepatic PM Ca(2+)-ATPase activity after 4 hours of storage and near-total inhibition after 12 hours. Porcine hepatic ER Ca(2+)-ATPase activity showed a 45% decrease in activity by 12 hours and a 69% decrease by 18 hours. We conclude that cold ischemia at clinically relevant times depresses PM Ca(2+)-ATPase more than ER Ca(2+)-ATPase activity in pig liver homogenates.

Expression of Intracellular Calcium Channels and Pumps after Partial Hepatectomy in Rat

Ca2+ signals regulate many cellular functions, including proliferation. They are governed by the inositol 1,4,5-trisphosphate receptor (IP3R), the only intracellular hepatic Ca2+ channel and by the endoplasmic reticulum Ca2+ pumps, SERCA. To characterise their role in regeneration, expression of their isoforms was studied after 2/3 hepatectomy by real-time quantitative PCR, Western blot and binding studies. We found an early increase in the expression of the IP3R isoform 1 which contrasted with the decrease of the expression of the IP3R isoforms 2 and 3 and of SERCA3. This results in a transient switch between IP3R isoforms 1 and 2, IP3R isoform 1 becoming predominant before the first round of mitosis. Binding studies detected a 30% diminution of the IP3R population at 24 h. In conclusion, the Ca2+ signalling machinery is regulated, after hepatectomy, by changes in expression of the IP3R and SERCA isoforms to adapt Ca2+ signals to the regenerative state.

Expression of inositol 1,4,5-trisphosphate receptor isoforms in rat cirrhosis.

Ca(2+) signals mediate the hepatic effects of numerous hormones and growth factors. Hepatic Ca(2+) signals are elicited by the inositol trisphosphate receptor, an intracellular Ca(2+) channel. Three isoforms of this receptor have been identified; they are expressed and regulated differently. We investigated the effect of liver fibrosis and cirrhosis on the hepatic expression of the inositol trisphosphate receptor isoforms. Two different rat models were used: bile duct ligation (fibrosis) and chronic exposure to CCl(4)/phenobarbital (cirrhosis). Messenger RNA levels were determined by ribonuclease protection assay (RPA), competitive polymerase chain reaction (PCR) followed by Southern blotting, and real-time quantitative PCR. Protein expression was assessed by Western blotting; tissue distribution was assessed by immunohistology. In control animals, isoform 2 was the predominant isoform, isoform 1 represented less than one third, and isoform 3 less than 1%. After bile duct ligation, expression of types 1 and 3 increased 1.9- and 5.7-fold, and expression of type 2 decreased 2. 5-fold at the protein level. After exposure to CCl(4)/phenobarbital, expression of types 1, 2, and 3 were 2.4-, 0.9-, and 4.2-fold their expression in control animals. Type 2 was localized to the apical domain of hepatocytes, consistent with a role for Ca(2+) signals in canalicular function. Type 3 was detectable in intrahepatic bile duct epithelial cells and not in hepatocytes, suggesting that Ca(2+) signals may be regulated differently in these cells. Signaling through inositol trisphosphate receptor participates in the pathogenesis of cirrhosis, because this process affects the expression of its isoforms.

Expression of calcium-binding protein regucalcin mRNA in the cloned rat hepatoma cells (H4-II-E) is stimulated through Ca2+ signaling factors: involvement of protein kinase C.

The expression of hepatic Ca2+-binding protein regucalcin in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in H4-II-E hepatoma cells. This expression was clearly stimulated in the presence of serum (10% fetal bovine serum). Bay K 8644 (2. 5 x 10(-6) M), a Ca2+ channel agonist, significantly stimulated regucalcin mRNA expression in the absence or presence of 10% serum. Dibutyryl cyclic AMP (10(-3) M) did not have a stimulatory effect on the regucalcin mRNA expression. The presence of phorbol 12-myristate 13-acetate (PMA; 10(-6) M) or estrogen (10(-8) M) caused a significant increase in regucalcin mRNA levels in the hepatoma cells cultured in serum-free medium, while insulin (5 x 10(-9) M) or dexamethasone (10(-6) M) had no effect. Bay K 8644-stimulated regucalcin mRNA expression in the hepatoma cells was completely blocked in the presence of trifluoperazine (10(-5) M), an antagonist of calmodulin, or staurosporine (10(-7) M), an inhibitor of protein kinase C. The stimulatory effect of PMA was clearly inhibited in the presence of stauroporine. The present study demonstrates that regucalcin mRNA is expressed in the transformed H4-II-E hepatoma cells, and that the expression is stimulated through Ca2+-dependent signaling factors.

Modifications of Intracellular Calcium Release Channels and Calcium Mobilization Following 70% Hepatectomy

The aim of this study was to investigate the properties of ryanodine and IP3receptors in regenerating liver following 70% hepatectomy, and to evaluate the hepatic Ca2+distribution and mobilization during this process. Specific [3H]ryanodine and [3H]IP3binding to hepatic smooth endoplasmic reticulum membranes, as well as subcellular Ca2+determination by atomic absorption flame photometry and Ca2+mobilization by INDO-1 AM spectrofluorescence in hepatocytes, was performed in regenerating livers after surgical 70% hepatectomy. Incorporation of14C amino acids into proteins and of32P into phospholipids was done in subcellular fractions. Ryanodine receptorKdpresented a dramatic increase after 12 h of surgery and remained high up to 2 days of treatment. IP3receptorBmaxshowed a significant augmentation starting at 6 h after hepatectomy and returning to normal values after 1 week. Cytosolic total calcium content decreased from 12 h until 4 days after hepatectomy whereas the microsomal and mitochondrial total calcium increased at 1 and 2–4 days of liver regeneration, which coincided with the differential turnover of proteins and phospholipids in these fractions. ATP-induced Ca2+transients in hepatocytes of 24-h-hepatectomized rats confirmed the altered sensitivity of the ryanodine receptor toward its ligand, since 10 times more ryanodine was necessary to alter the ATP-induced Ca2+transient. The data support the notion that the calcium release channels are targets of mechanisms of metabolic control during the proliferative response following 70% hepatectomy and might be part of the modified intracellular Ca2+dynamics during liver regeneration.

812 - Inhibitory effect of radiosensltizer AK-2123 on experimental hepatic metastases and CA 2+ active transport

812 - Inhibitory effect of radiosensltizer AK-2123 on experimental hepatic metastases and CA 2+ active transport