BBA - Molecular Basis of Disease (v.1500, #2)

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture by Maria C. Martinez-Diez; Maria A. Serrano; Maria J. Monte; Jose J.G. Marin (153-160).
Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose–response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species – cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10–1000 μM – revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (−50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause–effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.
Keywords: Liver; Hepatocyte culture; Toxicity; Neural red; Formazan; DNA synthesis;

Fructose-mediated damage to lens α-crystallin: prevention by pyruvate by Wei Zhao; Palaniyandi S Devamanoharan; Shambhu D Varma (161-168).
Post-translational modifications in lens crystallins due to glycation and oxidation have been suggested to play a significant role in the development of cataracts associated with aging and diabetes. We have previously shown that α-keto acids, like pyruvate, can protect the lens against oxidation. We hypothesize that they can also prevent the glycation of proteins competitively by forming a Schiff base between their free keto groups and the free –NH2 groups of protein as well as subsequently inhibit the oxidative conversion of the initial glycation product to advanced glycation end products (AGE). The purpose of this study was to investigate these possibilities using purified crystallins. The crystallins isolated from bovine lenses were incubated with fructose in the absence and presence of pyruvate. The post-incubation mixtures were analyzed for fructose binding to the crystallins, AGE formation, and the generation of high molecular weight (HMW) proteins. In parallel experiments, the keto acid was replaced by catalase, superoxide dismutase (SOD), or diethylene triaminepentaacetic acid (DTPA). This was done to ascertain oxidative mode of pyruvate effects. Interestingly, the glycation and consequent formation of AGE from α-crystallin was more pronounced than from β-, and γ-crystallins. The changes in the crystallins brought about by incubation with fructose were prevented by pyruvate. Catalase, SOD, and DTPA were also effective. The results suggest that pyruvate prevents against fructose-mediated changes by inhibiting the initial glycation reaction as well as the conversion of the initial glycated product to AGE. Hence it is effective in early as well as late phases of the reactions associated with the formation of HMW crystallin aggregates.
Keywords: Lens crystallin; Fructose; Glycation; Oxygen radical; Cataract; Pyruvate;

Increased Na+/H+ exchanger isoform 1 activity in spontaneously hypertensive rats: lack of mutations within the coding region of NHE1 by Sergei N. Orlov; Viacheslav A. Adarichev; Alison M. Devlin; Nathalie V. Maximova; Yu-Lin Sun; Johanne Tremblay; Anna F. Dominiczak; Yuvenali V. Postnov; Pavel Hamet (169-180).
Enhanced Na+/H+ exchange, measured as amiloride derivative-sensitive Na+ and H+ fluxes in cells with a preliminary acidified cytoplasm (Δμ H+-induced Na+/H+ exchange), is one of the most prominent intermediate phenotypes of altered vascular smooth muscle cell (VSMC) function in spontaneously hypertensive rats (SHR). Analysis of Na+/H+ exchange in F2 hybrids of SHR and normotensive rats seems to be the most appropriate approach in the search for the genetic determinants of abnormal activity of this carrier. However, the measurement of Δμ H+-induced Na+/H+ exchange is hardly appropriate for precise analysis of the carrier’s activity in VSMC derived from several hundred F2 hybrids. To overcome this problem, we compared the rate of 22Na influx under baseline conditions and in Na+-loaded (ouabain-treated) VSMC. The dose-dependency of the rate of Δμ H+-induced H+ efflux as well as of 22Na influx in control and ouabain-treated cells on ethylisopropylamiloride (EIPA) concentration were not different (K 0.5∼0.3 μM), suggesting that these ion transport pathways are mediated by the same carrier. EIPA-sensitive 22Na influx in Na+-loaded cells was ∼6-fold higher than in ouabain-untreated VSMC and was increased by 50–70% in two different substrains of SHR. About the same increment of EIPA-sensitive 22Na influx in Na+-loaded VSMC was observed in 5- to 6-week-old SHR (an age at which hypertension has not yet developed) as well as in stroke-prone SHR (SHRSP) with severe hypertension, indicating that the heightened activity of Na+/H+ exchange is not a consequence of long-term blood pressure elevation. To examine whether or not the augmented activity of Na+/H+ exchange in SHR is caused by mutation of NHE1, i.e. the only isoform of this carrier expressed in VSMC, we undertook single-stranded conformational polymorphism analysis of 23 NHE1 cDNA fragments from SHR and SHRSP and sequencing of the 456–2421 NHE1 cDNA fragment. This study did not reveal any mutation in the entire coding region of NHE1. The lack of mutation in the coding region of NHE1 indicates that the augmented activity of the ubiquitous Na+/H+ exchanger in primary hypertension is caused by altered regulation of carrier turnover number or/and its plasma membrane content.
Keywords: Na+/H+ exchange; Vascular smooth muscle; Spontaneous hypertension;

Evaluation of oxidative stress based on lipid hydroperoxide, vitamin C and vitamin E during apoptosis and necrosis caused by thioacetamide in rat liver by Fang Sun; Shoko Hayami; Yukako Ogiri; Sakiko Haruna; Kyoko Tanaka; Yasuko Yamada; Sadako Tokumaru; Shosuke Kojo (181-185).
After 12 h of thioacetamide (500 mg/kg body weight) administration to rats, the activity of caspase-3-like protease in the liver increased significantly compared to that in the control group. In plasma, the activity of caspase-3 was barely detectable in the control rat, but had increased significantly after 24 h of drug administration along with a dramatic increase in GOT. These results indicate that thioacetamide causes apoptosis in the liver by activating caspase-3, which is released to plasma by successive necrosis. At 24 h, the concentration of liver lipid hydroperoxides, a mediator of radical reaction, was 2.2 times as high as that of control rats. After 12 and 24 h of thioacetamide administration, the liver concentrations of vitamins C and E decreased significantly. The decrease of antioxidants and formation of lipid hydroperoxides 24 h after thioacetamide administration support the view that extensive radical reactions occur in the liver during the necrotic process.
Keywords: Thioacetamide; Vitamin C; Vitamin E; Hydroperoxide; Necrosis; Apoptosis;

Low-frequency low-field magnetic susceptibility of ferritin and hemosiderin by P.D. Allen; T.G. St Pierre; W. Chua-anusorn; V. Ström; K.V. Rao (186-196).
Low-frequency low-field magnetic susceptibility measurements were made on four samples of mammalian tissue iron oxide deposits. The samples comprised: (1) horse spleen ferritin; (2) dugong liver hemosiderin; (3) thalassemic human spleen ferritin; and (4) crude thalassemic human spleen hemosiderin. These samples were chosen because Mössbauer spectroscopic measurements on the samples indicated that they exemplified the variation in magnetic and mineral structure found in mammalian tissue iron oxide deposits. The AC-magnetic susceptometry yielded information on the magnetization kinetics of the four samples indicating samples 1, 2, and 3 to be superparamagnetic with values of around 1011 s−1 for the pre-exponential frequency factor in the Néel–Arrhenius equation and values for characteristic magnetic anisotropy energy barriers in the range 250–400 K. Sample 4 was indicated to be paramagnetic at all temperatures above 1.3 K. The AC-magnetic susceptometry data also indicated a larger magnetic anisotropy energy distribution in the dugong liver sample compared with samples 1 and 3 in agreement with previous Mössbauer spectroscopic data on these samples. At temperatures below 200 K, samples 1–3 exhibited Curie–Weiss law behavior, indicating weak particle–particle interactions tending to favor antiparallel alignment of the particle magnetic moments. These interactions were strongest for the dugong liver hemosiderin, possibly reflecting the smaller separation between mineral particles in this sample. This is the first magnetic susceptometry study of hemosiderin iron deposits and demonstrates that the AC-magnetic susceptometry technique is a fast and informative method of studying such tissue iron oxide deposits.
Keywords: AC-magnetic susceptibility; Ferritin; Hemosiderin; Superparamagnetism; Iron oxide; Mössbauer spectroscopy;

A male child, who presented at the age of 3.5 years with acute renal failure, was diagnosed as having partial deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC The underlying HPRT mutation was unique in that the specific activity of HPRT in erythrocyte and in fibroblast lysates was normal, but the rate of uptake of hypoxanthine into nucleotides of intact cultured fibroblasts was markedly reduced (23% of normal). The low functioning of HPRT in the intact fibroblasts was associated with decreased utilization of endogenously generated hypoxanthine and with decreased utilization of the cosubstrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The non-utilized hypoxanthine was excreted into the incubation medium. The accumulation of PRPP was indicated by the 2.3-fold increase in the rate of uptake of adenine into intact cell nucleotides and by the 7.5-fold enhancement of the rate of de novo purine synthesis. Kinetic studies of HPRT activity in fibroblast lysates revealed reduced affinity of the enzyme for PRPP (apparent K m 500 μM in comparison to 25 μM in control lysates), manifested in low activity at low (physiological), but not at high PRPP concentrations. The apparent K m for hypoxanthine was normal (23 μM in comparison to 14.2 μM in control lysates). With allopurinol treatment, our patient has had no problems since presentation, and is developing normally at 5 years of age.
Keywords: Hypoxanthine-guanine phosphoribosyltransferase deficiency; Kelley-Seegmiller syndrome; Lesch-Nyhan syndrome;

Attenuation of intestinal ischemia/reperfusion injury with sodium nitroprusside: studies on mitochondrial function and lipid changes by Muniswamy Madesh; Anup Ramachandran; Anna Pulimood; Markandeyulu Vadranam; K.A Balasubramanian (204-216).
Reactive oxygen species have been implicated in cellular injury during ischemia/reperfusion (I/R). Mitochondria are one of the main targets of oxygen free radicals and damage to this organelle leads to cell death. Reports suggest that nitric oxide (NO) may offer protection from damage during I/R. This study has looked at the functional changes and lipid alteration to mitochondria during intestinal I/R and the protection offered by NO. It was observed that I/R of the intestine is associated with functional alterations in the mitochondria as suggested by MTT reduction, respiratory control ratio and mitochondrial swelling. Mitochondrial lipid changes suggestive of activation of phospholipase A2 and phospholipase D were also seen after (I/R) mediated injury. These changes were prevented by the simultaneous presence of a NO donor in the lumen of the intestine. These studies have suggested that structural and functional alterations of mitochondria are prominent features of I/R injury to the intestine which can be ameliorated by NO.
Keywords: Intestine; Ischemia-reperfusion; Nitric oxide; Mitochondria; Phospholipase;

Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation by Jian-Gang Shen; Xing-Sheng Quo; Bo Jiang; Ming Li; Wen-juan Xin; Bao-Lu Zhao (217-226).
The inhibitory effects of Chinonin, a natural antioxidant extracted from a Chinese medicine, on apoptotic and necrotic cell death of cardiomyocytes in hypoxia-reoxygenation process were observed in this study. The possible mechanisms of Chinonin on scavenging reactive oxygen species and regulating apoptotic related genes bcl-2 and p53 were also investigated. Neonatal rat cardiomyocytes were subjected to 24-h hypoxia and 4-h reoxygenation. Cell death was evaluated by DNA electrophoresis on agarose gel, cell death ELISA and annexin-V-FLUOS/propidium iodide (PI) double staining cytometry. Hypoxia caused the increase of apoptotic rates and the release of lactate dehydrogenase (LDH), while reoxygenation not only further increased the apoptotic rates and leakage of LDH, but also induced necrosis of cardiomyocytes. In addition, hypoxia increased the levels of NO2 /NO3 and thiobarbituric acid reacted substances (TBARS), while reoxygenation decreased NO2 /NO3 , but further increased TBARS in the cultured media. Moreover, hypoxia up-regulated the expression levels of bcl-2 and p53 proteins, while reoxygenation down-regulated bcl-2 and further up-regulated p53. Chinonin significantly decreased the rates of apoptotic and necrotic cardiomyocytes, and inhibited the leakage of LDH. It also diminished NO2 /NO3 and TBARS, down-regulated the expression level of p53 protein, and up-regulated bcl-2 protein, respectively. The results suggest that Chinonin has preventive effects against apoptotic and necrotic cell death and its protective mechanisms are related to the antioxidant properties of scavenging nitric oxide and oxygen free radicals, and the modulating effects on the expression levels of bcl-2 and p53 proteins.
Keywords: Chinonin; Apoptosis; Nitric oxide; Oxygen free radicals; p53; bcl-2; Hypoxia; Reoxygenation;

CXCR4 on human endothelial cells can serve as both a mediator of biological responses and as a receptor for HIV-2 by Marina Molino; Marilyn J. Woolkalis; Nicolas Prevost; Domenico Praticó; Elliot S. Barnathan; Giulia Taraboletti; Beth Stobenau Haggarty; Joseph Hesselgesser; Richard Horuk; James A. Hoxie; Lawrence F. Brass (227-240).
It has been shown that deletion of the chemokine receptor, CXCR4, causes disordered angiogenesis in mouse models. In the present studies, we examined the distribution and trafficking of CXCR4 in human endothelial cells, tested their responses to the CXCR4 ligand, SDF-1, and asked whether endothelial cell CXCR4 can serve as a cell surface receptor for the binding of viruses. The results show that CXCR4 is present on endothelial cells from coronary arteries, iliac arteries and umbilical veins (HUVEC), but expression was heterogeneous, with some cells expressing CXCR4 on their surface, while others did not. Addition of SDF-1 caused a rapid decrease in CXCR4 surface expression. It also caused CXCR4-mediated activation of MAPK, release of PGI2, endothelial migration, and the formation of capillary-like structures by endothelial cells in culture. Co-culture of HUVEC with lymphoid cells that were chronically infected with a CD4-independent/CXCR4-tropic variant of HIV-2 resulted in the formation of multinucleated syncytia. Formation of the syncytia was inhibited by each of several different CXCR4 antibodies. Thus, our findings indicate: (1) that CXCR4 is widely expressed on human endothelial cells; (2) the CXCR4 ligand, SDF-1, can evoke a wide variety of responses from human endothelial cells; and (3) CXCR4 on endothelial cells can serve as a receptor for isolates of HIV that can utilize chemokine receptors in the absence of CD4.
Keywords: Endothelial cell; Chemokine; Chemokine receptor; HIV-2; CXCR4; SDF-1;

Interleukin-1β regulates CFTR expression in human intestinal T84 cells by Eduardo G. Cafferata; Anatilde M. González-Guerrico; Luciana Giordano; Omar H. Pivetta; Tomás A. Santa-Coloma (241-248).
Cystic fibrosis is an autosomal recessive genetic disease, produced by a mutation in the CFTR gene that impairs its function as a chloride channel. In this work, we have examined the effects of interleukin-1β (IL-1β) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1β (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1β (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 μM) and GF109203X (1 μM) inhibited the stimulatory effect of IL-1β. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 μM) and herbymicin A (2 μM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1β. The repression of CFTR up-regulation by cycloheximide (35.5 μM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 μM), suggest that the increased mRNA levels seen after IL-1β treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1β, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.
Keywords: Interleukin-1β; CFTR; Cystic fibrosis;

Right ventricular upregulation of the Ca2+ binding protein S100A1 in chronic pulmonary hypertension by Philipp Ehlermann; Andrew Remppis; Oliver Guddat; Jörg Weimann; Philipp A. Schnabel; Johann Motsch; Claus W. Heizmann; Hugo A. Katus (249-255).
The Ca2+ binding protein S100A1 increases the Ca2+ release from the sarcoplasmatic reticulum by interacting with the ryanodine receptor. In order to understand whether this effect might be operative in the early course of hypertrophy, when myocardium is able to meet increased workload, we investigated the expression of S100A1 in a model of moderate right ventricular hypertrophy. The pulmonary arteries of nine pigs were embolised three times with Sephadex G-50. After 70 days, all pigs showed a moderate pulmonary hypertension. Right ventricular tissue of embolised animals showed a significant increase of connective tissue and enlargement of myocyte diameters. In controls, we found a differential expression of S100A1 with significantly lower S100A1 protein levels in right ventricular compared to left ventricular tissue. In pulmonary hypertension, S100A1 expression increased significantly in hypertrophied right ventricles while it was unchanged in left ventricular tissue. No change was observed in the expression of SERCA2a and phospholamban. Our data show, for the first time, that moderate pressure overload results in an upregulation of S100A1. This may reflect an adaptive response of myocardial Ca2+ homeostasis to a higher workload.
Keywords: Calcium-binding protein; S100A1; Myocardial hypertrophy; Pulmonary hypertension; SERCA; Phospholamban;