Aging is associated with increased clonogenic potential in rat liver in vivo

Cancer increases with age and often arises from the selective clonal growth of altered cells. Thus, any environment favoring clonal growth per se poses a higher risk for cancer development. Using a genetically tagged animal model, we investigated whether aging is associated with increased clonogenic potential. Groups of 4-, 12-, 18-, and 24-month-old Fischer 344 rats were infused (via the portal vein) with 2x10(6) hepatocytes isolated from a normal syngenic 2-month-old donor. Animals deficient in dipeptidyl-peptidase type IV (DPP-IV-) enzyme were used as recipients, allowing for the histochemical detection of injected DPP-IV+ cells. Groups of animals were sacrificed at various times thereafter. No growth of DPP-IV+ transplanted hepatocytes was present after either 2 or 6 months in the liver of rats transplanted at young age, as expected. In striking contrast, significant expansion of donor-derived cells was seen in animals transplanted at the age of 18 months: clusters comprising 7-10 DPP-IV+ hepatocytes/cross-section were present after 2 months and were markedly enlarged after 6 months (mean of 88+/-35 cells/cluster/cross-section). These results indicate that the microenvironment of the aged liver supports the clonal expansion of transplanted normal hepatocytes. Such clonogenic environments can foster the selective growth of pre-existing altered cells, thereby increasing the overall risk for cancer development associated with aging.     

Effect of ecdysterone on glucose metabolism in vitro

The aims of this study was to investigate whether ecdysterone is able to exert glucose-lowering effect on hepatocytes or stimulate the secretion of insulin. HepG2 cell line was used for glucose consumption (GC) studies. At moderate high glucose concentration (11.1 mmol/L), GC of HepG2 cells was increased by 44% to 77% with ecdysterone 1 x 10(-6) to 1 x 10(-4) mol/L, which was comparable to that with 1 x 10(-3) mol/L metformin. The glucose-lowering effect of ecdysterone decreased as the glucose concentration of medium increased. The maximal potency was reached in the presence of 5.5 mmol/L glucose, and the effect was disappeared as the glucose consumption was increased to 22.2 mmol/L. This effect was independent on insulin concentration, which was similar to that of metformin and was different from that of troglitazone, whose glucose-lowering effect was insulin-dependent. Troglitazone had a better antihyperglycemic potency than metformin when insulin was added. Simultaneously, a significant toxicity of troglitazone to HepG2 cells was observed. betaTC3 cells were not stimulated by ecdysterone, that is, no secretogogue effect of ecdysterone was observed. The results indicate that ecdysterone is able to exert the glucose-lowering effect in hepatocytes which is insulin-independent, but has no effect on insulin release.     

Platelet-activating factor increases inositol phosphate production and cytosolic free Ca2+ concentrations in cultured rat Kupffer cells

Platelet-activating factor (PAF) stimulates glycogenolysis in perfused livers but not in isolated hepatocytes [(1984) J. Biol. Chem. 259, 8685-8688]. PAF-induced glycogenolysis in liver is associated closely with a pronounced constriction of the hepatic vasculature [(1986) J. Biol. Chem. 261, 644-649]. These and other observations suggest that PAF stimulates glycogenolysis in liver indirectly by interactions with cells other than hepatocytes. We have evaluated effects of PAF on hepatic Kupffer cells, which regulate flow through the hepatic sinusoids. Application of PAF to [3H]inositol-labeled Kupffer cells produced dose-dependent increases in [3H]inositol phosphates with an EC50 value of 4 x 10(-10) M. Increases in inositol phosphate production in response to PAF were inhibited by a specific PAF receptor antagonist, SRI 63-675 (2 x 10(-7) M), and stimulus of protein kinase C, phorbol 12-myristate 13-acetate (1 x 10(-7) M). Measurements of cytosolic free Ca2+ concentrations ([Ca2+]i) in single Kupffer cells loaded with Fura-2 demonstrated that application of PAF (2 x 10(-9) M) resulted in significant increases in [Ca2+]i. These observations lead us to propose that interactions of PAF with Kupffer cells may result in the hemodynamic and metabolic responses to PAF in liver.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

小鼠LAK细胞对粒—单系祖细胞增殖的影响

Murine lymphokine-activated killer (LAK) cells were generated from spleen cells of C57/BL6 mice by culture of spleen cells in vitro for 72 hours in medium containing 500 units/ml recombinant human interleukin 2 (IL-2), and effects of these LAK cells on proliferation of syngenic myeloid progenitor cells (CFU-GM) were observed. After 3 days culture, LAK cells were assayed for their cytotoxicity in a 4 hours 51Cr-release test. Either natural killer (NK) cell sensitive YAC-1 lymphoma cells or NK cell resistant LP-3 and WEHI-164 fibrosarcoma cells were efficiently lysed by murine LAK cells. When LAK cells were added into culture system in a final concentration of 5 x 10(4)/ml, 2 x 10(5)/ml, 8 x 10(5)/ml, CFU-GM were increased by 55.2%, 165.5%, and 194.4% of control respectively. LAK-CM also showed augmentative effect on CFU-GM growth. When 10% (v/v) of LAK-CM were added into culture system, CFU-GM were increased by 51.4% of control, but LAK-CM alone could not stimulate CFU-GM growth. Again, effects of LAK-BMC interaction on CFU-GM formation were investigated. CFU-GM were inhibited to 27.6% of control when 1 x 10(5) BMC were mixed with 8 x 10(5) LAK cells and incubated for 4 hours prior to CFU-GM culture. These data suggest that (1) LAK cells may secrete co-CSF which showed synergistic effect with CSF on CFU-GM proliferation: (2) When LAK cells contact with BMC, they showed significant cytotoxicity to myeloid progenitor cells which mediated decrease of CFU-GM formation.     

人胎肝中肝细胞生长因子生物活性的研究

人胎肝细胞裂解液经膜超滤,在分子量10～30kD组分中可检测出人肝细胞生长因子(hHGF)活性。hHGF为一热稳定的蛋白质或多肽类物质。它可特异地刺激肝来源细胞~3H-TdR掺入的增加,并且存在量效依赖关系,而对非肝来源细胞的DNA合成无刺激作用。hHGF的生物活性及理化性质与某些已知因子,如胰岛素、胰高血糖素、血小板来源的生长因子、表皮生长因子及增殖刺激因子等有所不同。     

Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.     

Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo

Although human amniotic fluid is an attractive source of multipotent stem cells, the potential of amniotic fluid stem cells (AFSCs) to differentiate into hepatic cells has not been extensively evaluated. In this study, we examined whether human AFSCs can differentiate into a hepatic cell lineage in vitro and in vivo. After being treated with cytokines (fibroblast growth factor 4, basic fibroblast growth factor, hepatocyte growth factor, and oncostatin), AFSCs developed a morphology similar to that of hepatocytes. RT-PCR and immunofluorescence analysis showed that the treated AFSCs expressed the hepatocyte-specific markers albumin, cytokeratin 18, and alpha-fetoprotein. The differentiated cells also developed hepatocyte-specific functions, i.e., they secreted albumin, absorbed indocyanine green, and stored glycogen. When transplanted into CCl(4)-injured immunodeficient mice, undifferentiated AFSCs were integrated into the liver tissue, and they expressed markers characteristic of mature human hepatocytes. Although integration of AFSCs into the liver was limited (0.1-0.3% of hepatocytes), histological analysis showed that the recipient mice recovered more rapidly from CCl(4) injury than CCl(4)-injured mice that did not receive AFSCs. AFSCs can differentiate into hepatocyte-like cells in vitro and in vivo and can represent an easily accessible source of progenitor cells for hepatocyte regeneration and liver cell transplantation.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

DNA ploidy and autophagic protein degradation as determinants of hepatocellular growth and survival

Hepatocytes have the ability to go through specialized cell cycles, which, during normal developmental liver growth, result in the formation of binuclear and polyploid cells. In the adult rat liver, the majority of the hepatocytes (about 70%) are tetraploid, 15-20% are octoploid, and only 10-15% are diploid (about 50% in humans). One-third of the hepatocytes in either rats or humans are binuclear (with two diploid or two tetraploid nuclei). Among cultured rat hepatocytes stimulated with growth factors (EGF and insulin), one-half of the mitoses are of the binucleating type (suggesting a "quantal" mechanism), causing one-third of the postmitotic cells to become binuclear. In contrast, regenerative liver growth, induced by partial hepatectomy, is predominantly nonbinucleating. During rat liver carcinogenesis, the early populations of phenotypically altered cells (foci) are predominantly diploid, as are the later neoplastic nodules and carcinomas, which can be shown to have a regeneration-like, largely nonbinucleating growth pattern. A negative correlation between growth capacity and ploidy can be demonstrated in cultured hepatocytes, regenerating livers, neoplastic nodules, and hepatocellular carcinomas, suggesting that suppression of binucleation and polyploidization may carry a growth advantage, in addition to helping to maintain a large population of diploid, potential stem cells. Since a diploid genome is less protected against mutagenic change than a polyploid genome, diploid tumor cells may, furthermore, be more prone than polyploid cells to undergo mutation-based progression toward increasing malignancy. The ability of liver tumor promoters like 2-acetylaminofluorene, cyproterone acetate, -hexachlorocyclohexane and methylclofenapate to induce nonbinucleating hepatocyte growth may, therefore, cooperate with the selective growth stimulation of cancer cells and cancer cell precursors to promote liver carcinogenesis.Autophagy, a mechanism for the bulk degradation of cytoplasm, contributes to intracellular protein turnover and serves to restrict cellular growth. Rat liver carcinogenesis is accompanied by a progressive reduction of autophagic capacity, preneoplastic livers having 50% and hepatocellular carcinoma cells only 20% as much autophagy as normal hepatocytes. The ascites hepatoma cell line AH-130 has virtually no autophagy during logarithmic growth, but some autophagy is turned on when the cells become growth-arrested at high cell density. Ascitic fluid from AH-130 cells is able to completely inhibit autophagy in normal hepatocytes, suggesting that the cancer cells may improve their growth ability through an autocrine, autophagy-suppressive mechanism. Hepatocytes from preneoplastic livers similarly maintain a low autophagic activity under restrictive culture conditions, thereby surviving much better than normal hepatocytes, which switch on their autophagy. In the presence of an autophagy inhibitor (3-methyladenine), normal and preneoplastic hepatocytes survive equally well, testifying to the importance of autophagy as a determinant of cell survival and growth.     

Involvement of CXCR3-associated chemokines in MHV-3 induced fulminant hepatic failure

The role of chemokines in murine hepatitis virus strain 3 (MHV-3) induced fulminant hepatic failure (FHF) is not well defined. In this study, we investigated the role of the CXC chemokine receptor 3 (CXCR3)-associated chemokine [monokine induced by IFN-gamma (Mig/CXCL9) and interferon-gamma-inducible protein 10 (IP-10/CXCL10)] in the recruitment of intrahepatic lymphocytes and subsequent fulminant hepatic failure induced by MHV-3. Balb/cJ mice (6–8 weeks, female) were intraperitioneally injected with 100 PFU MHV-3.The proportions and numbers of T cells and NK cells as well as the expression of CXCR3 on T cells and NK cells in the liver, spleen and blood were analyzed by flow cytometry. The hepatic mRNA level of the CXCR3-associated chemokines (CXCL9 and CXCL10) was detected by realtime PCR. A transwell migration assay was used to assess the chemotactic effect of MHV-3-infected hepatocytes on the splenic lymphocytes. Following MHV-3 infection, the number of hepatic NK cells and T cells and the frequencies of hepatic NK cells and T cells expressing CXCR3 increased markedly; however, in the spleen and peripheral blood, they both decreased significantly. Moreover, the hepatic mRNAs levels of CXCL9 and CXCL10 were significantly elevated post infection. The transwell migration assay demonstrated that MHV-3-infected hepatocytes have the capacity to attract and recruit the splenic NK cells and T cells, and CXCL10 plays a key role in lymphocyte mobilization from the spleen. These results suggest that the CXCR3-associated chemokines (CXCL9 and CXCL10) may play an important role in the recruitment of intrahepatic lymphocytes and subsequent necroinflammation and hepatic failure in MHV-3 infection.     

Stimulation by N6,O2'-dibutyrul andenosine 3',5'-cyclic monophosphate of RNA and DNA synthesis and of cell proliferation of rat hepatocytes in primary tissue culture.

The effects of DBcAMP in doses from 1.5 x 10(-8) to 1.5 x 10(-3) M on the compartmental apparent surface area (ASA) and (5(-3H)uridine radioactivity concentration (URC), (methyl-3H)thymidine labelling index per 1 hour ([Me-3H]Tdr LI/h) and per cent mitotic index (MI%) and colchicine metaphase index (CMI%) of young rat differentiated hepatocytes in primary tissue culture were investigated by morphometric and radioautographie methods. In these cells DBcAMP was found to elicit: (1) progressive increments in the ASA of nucleoli, karyoplasm and cytoplasm; (2) peak increases in nucleolar URC at 1.5 x 10(-8) and 10(-5) M, but a slight decrease at 1.5 x 10(-3) M; (3) singificant increments in karyoplasmic and total nuclear URC at all doses, except at 1.5 x 10(-6) and 10(-4) M, when such parameters remained at control levels; (4) steady and progressive increases in cytoplasmic and total cell URC values; (5) marked increments in (Me-3H)Tdr LI/h, MI% and CMI% up to the dose of 1.5 x 10(-4) M, but at 1.5 x 10(-3) M these parameters were found to be either much less enhanced or to approach closely to control values. cAMP in doses from 1.5 x 10(-8) to 10(-4) M also markedly incremented the in vitro hepatocyte CMI%, while having a lesser stimulatory effect at 1.5 x 10(-3)M. Finally of the various possible metabolites of DBcAMP administered at 1.5 x 10(-8) M to liver cultures, N6- and O2'-MBcAMP and, again, cAMP significantly increased the CMI%, of cultured hepatocytes, whereas 5'-AMP, adenosine and allantoin had no significant effect and Na-butyrate slightly decreased it. The present observations strengthen the hypothesis that cAMP and its butyrated derivatives, by possibly amplifying the template activity of the liver chromatin, accelerate the flow of differentiated primary young rat hepatocytes into the various stages of the mitotic cell cycle.     

The magnetic susceptibilities of iron deposits in thalassaemic spleen tissue

The iron-specific magnetic susceptibility of tissue iron deposits is used in the field of non-invasive measurement of tissue iron concentrations. It has generally been assumed to be a constant for all tissue and disease types. The iron-specific magnetic susceptibilities chi(Fe) for spleen tissue samples from 7 transfusion dependent beta-thalassaemia (beta-thal) patients and 11 non-transfusion dependent beta-thalassaemia/Haemoglobin E (beta/E) patients were measured at 37 degrees C. Both groups of patients were iron loaded with no significant difference in the distribution of spleen iron concentrations between the two groups. There was a significant difference between the mean chi(Fe) of the spleen tissue from each group. The non-transfusion dependent beta/E patients had a higher mean (+/-standard deviation) spleen chi(Fe) (1.55+/-0.23 x 10(-6) m(3)/kg Fe) than the transfusion dependent beta-thal patients (1.16+/-0.25 x 10(-6) m(3)/kg Fe). Correlations were observed between chi(Fe) of the spleen tissue and the fraction of magnetic hyperfine split sextet in the (57)Fe M?ssbauer spectra of the tissues at 78 K (Spearman rank order correlation r=-0.54, p=0.03) and between chi(Fe) of the spleen tissue and the fraction of doublet in the spectra at 5 K (r=0.58, p=0.02) indicating that chi(Fe) of the spleen tissue is related to the chemical speciation of the iron deposits in the tissue.     

Hepatogenic differentiation of mesenchymal stem cells in a rat model of thioacetamide-induced liver cirrhosis

Implantation of bone-marrow-derived MSCs (mesenchymal stem cells) has emerged as a potential treatment modality for liver failure, but in vivo differentiation of MSCs into functioning hepatocytes and its therapeutic effects have not yet been determined. We investigated MSC differentiation process in a rat model of TAA (thioacetamide)-induced liver cirrhosis. Male Sprague-Dawley rats were administered 0.04% TAA-containing water for 8 weeks, MSCs were injected into the spleen for transsplenic migration into the liver, and liver tissues were examined over 3 weeks. Ingestion of TAA for 8 weeks induced micronodular liver cirrhosis in 93% of rats. Injected MSCs were diffusely engrafted in the liver parenchyma, differentiated into CK19 (cytokeratin 19)- and thy1-positive oval cells and later into albumin-producing hepatocyte-like cells. MSC engraftment rate per slice was measured as 1.0-1.6%. MSC injection resulted in apoptosis of hepatic stellate cells and resultant resolution of fibrosis, but did not cause apoptosis of hepatocytes. Injection of MSCs treated with HGF (hepatocyte growth factor) in vitro for 2 weeks, which became CD90-negative and CK18-positive, resulted in chronological advancement of hepatogenic cellular differentiation by 2 weeks and decrease in anti-fibrotic activity. Early differentiation of MSCs to progenitor oval cells and hepatocytes results in various therapeutic effects, including repair of damaged hepatocytes, intracellular glycogen restoration and resolution of fibrosis. Thus, these results support that the in vivo hepatogenic differentiation of MSCs is related to the beneficial effects of MSCs rather than the differentiated hepatocytes themselves.     

Comparative study of oestrogenic properties of eight phytoestrogens in MCF7 human breast cancer cells

Previous studies have compared the oestrogenic properties of phytoestrogens in a wide variety of disparate assays. Since not all phytoestrogens have been tested in each assay, this makes inter-study comparisons and ranking oestrogenic potency difficult. In this report, we have compared the oestrogen agonist and antagonist activity of eight phytoestrogens (genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-prenylnaringenin, coumestrol and resveratrol) in a range of assays all based within the same receptor and cellular context of the MCF7 human breast cancer cell line. The relative binding of each phytoestrogen to oestrogen receptor (ER) of MCF7 cytosol was calculated from the molar excess needed for 50% inhibition of 3H]oestradiol binding (IC50), and was in the order coumestrol (35x)/8-prenylnaringenin (45x)/deoxymiroestrol (50x)>miroestrol (260x)>genistein (1000x)>equol (4000x)>daidzein (not achieved: 40% inhibition at 10(4)-fold molar excess)>resveratrol (not achieved: 10% inhibition at 10(5)-fold molar excess). For cell-based assays, the rank order of potency (estimated in terms of the concentration needed to achieve a response equivalent to 50% of that found with 17beta-oestradiol (IC50)) remained very similar for all the assays whether measuring ligand ability to induce a stably transfected oestrogen-responsive ERE-CAT reporter gene, cell growth in terms of proliferation rate after 7 days or cell growth in terms of saturation density after 14 days. The IC50 values for these three assays in order were for 17beta-oestradiol (1 x 10(-11)M, 1 x 10(-11)M, 2 x 10(-11)M), and in rank order of potency for the phytoestrogens, deoxymiroestrol (1 x 10(-10)M, 3 x 10(-11)M, 2 x 10(-11)M)>miroestrol (3 x 10(-10)M, 2 x 10(-10)M, 8 x 10(-11)M)>8-prenylnaringenin (1 x 10(-9)M, 3 x 10(-10)M, 3 x 10(-10)M)>coumestrol (3 x 10(-8)M, 2 x 10(-8)M, 3 x 10(-8)M)>genistein (4 x 10(-8)M, 2 x 10(-8)M, 1 x 10(-8)M)/equol (1 x 10(-7)M, 3 x 10(-8)M, 2 x 10(-8)M)>daidzein (3 x 10(-7)M, 2 x 10(-7)M, 4 x 10(-8)M)>resveratrol (4 x 10(-6)M, not achieved, not achieved). Despite using the same receptor context of the MCF7 cells, this rank order differed from that determined from receptor binding. The most marked difference was for coumestrol and 8-prenylnaringenin which both displayed a relatively potent ability to displace [3H]oestradiol from cytosolic ER compared with their much lower activity in the cell-based assays. Albeit at varying concentrations, seven of the eight phytoestrogens (all except resveratrol) gave similar maximal responses to that given by 17beta-oestradiol in cell-based assays which makes them full oestrogen agonists. We found no evidence for any oestrogen antagonist action of any of these phytoestrogens at concentrations of up to 10(-6)M on either reporter gene induction or on stimulation of cell growth.     

Amiloride and amiloride analogs inhibit Na+/K+-transporting ATPase and Na+-coupled alanine transport in rat hepatocytes

Amiloride, a commonly used inhibitor of Na+-H+ exchange, has been shown to exhibit a variety of nonspecific effects. Recently, the more potent amiloride analogs, 5-(N,N-dimethyl)amiloride hydrochloride (DMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIA), have been used to control for the nonspecific effects of the parent compound. In the present study, we have explored the effects of these analogs on Na+/K+-transporting ATPase (Na+/K+-ATPase) and Na+-coupled alanine transport in primary rat hepatocyte cultures and rat liver plasma membranes, and we have compared the effects of these analogs with the effects of amiloride and ouabain. Amiloride, DMA, and EIA increased steady-state Na+ content and inhibited ouabain-sensitive 86Rb+ uptake in a reversible, concentration-dependent, ouabain-like manner, with estimated 50% inhibitory concentrations (IC50) of 3.0.10(-3) M, 5.2.10(-4) M, and 1.2.10(-4) M, respectively. Amiloride, DMA and EIA also inhibited ouabain-sensitive ATP hydrolysis in rat liver plasma membranes with similar potency (IC50 values of 2.2.10(-3) M, 2.2.10(-3) M, and 1.7.10(-4) M, respectively). In separate experiments, amiloride (5.10(-3) M), DMA (10(-3) M), and EIA (2.5.10(-4) M) decreased the uptake into hepatocytes of alanine by 20%, 61%, and 59%, respectively, and further studies with DMA (10(-3) M) demonstrated that this inhibition was largely due to a decrease in the Na+-dependent fraction of alanine uptake. These findings indicate that amiloride, DMA, and EIA inhibit hepatic Na+/K+-ATPase directly, reversibly, and with a relative rank order potency of EIA greater than DMA greater than amiloride. All three compounds also inhibit the hepatic uptake of alanine, and presumably could indirectly inhibit other Na+-coupled transport processes as well.     

Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors

Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also manifest in pravastatin, is biochemically distinct from the pharmacodynamic-based disposition of lovastatin, which along with fluindostatin exhibited potent inhibition in all tissues that were exposed to it.     

Long term phenobarbital administration does not promote the multiplication of hepatocytes replicating after single cyproterone acetate administration

Cyproterone acetate (CPA) is a synthetic antiandrogenic compound which is widely used in clinic but suspected to be hepatocarcinogenic. CPA is also mitogenic in rat liver. Using genetic labeling of dividing cells, we examined whether hepatocytes dividing in response to acute CPA administration could give rise to preneoplastic foci after administration of a tumor promoter: phenobarbital. CPA was administered orally to rats and dividing hepatocytes were genetically labeled using retroviral vectors carrying the beta-galactosidase gene. After labeling rats were given phenobarbital for 10 months and sacrificed. The presence of beta-galactosidase labeled hepatocytes as well as preneoplastic hepatocytes was assessed by immunohistochemistry. Genetic labeling of hepatocytes was obtained in all animals. At the end of phenobarbital administration, no hepatic tumors were observed. Preneoplastic foci were not increased in treated animals as compared to control rats. Moreover beta-galactosidase positive hepatocytes were never detected in preneoplastic foci. Finally, the size of the beta-galactosidase positive clusters was smaller in treated animals as compared to control rats. We conclude that acute CPA administration is not carcinogenic in rat liver and does not initiate preneoplastic hepatocytes capable to give rise to foci after phenobarbital promotion. Therefore the mitogenic property of CPA is distinct from its putative carcinogenic activity. Finally, analysis of the size of beta-galactosidase positive cells clusters demonstrate that phenobarbital does not induce hepatocyte proliferation in rats.