Repair and replication of DNA in rat brain and liver during foetal and post-natal development,in relation to nitroso-alkyl-urea induced carcinogenesis

Summary The susceptibility of rat brain to induction of cancer by N-nitroso-N-ethyl-urea (NEU) increases dramatically from a very low level in the 12 day foetus to a maximum at the time of birth, and then decreases with age. Liver tumors are rarely induced by a single treatment with NEU at any age. If induction of cancer by nitroso-alkyl-ureas depends on replication of DNA containing the mispairing base O⁶-alkylguanine, susceptibility would reflect the balance between the protective effect of removal of the base by repair mechanisms and the potentiating effect of cell replication. The capacity of tissues to remove O⁶-alkylguanine from DNA depends on the amount of alkyl acceptor protein (AAP) present. To study the concept that carcinogenesis results from replication of alkylated DNA, the AAP contents and relative rates of DNA replication were studied in brain and liver of rats at various stages of development, from the 12 day foetus to the 48 week old rat. Replication in liver was approximately ten times higher than in intra-cranial contents at each stage of development studies. The AAP content was higher in the 12 day foetal brain than later, decreasing to low levels as susceptibility to NEU increased until the time of birth, and then remaining low in the adult. The peak sensitivity of brain therefore corresponds to the time at which AAP content is low and rate of DNA replication is high. With liver, AAP levels are low in the foetus, although higher than in brain, and increase after birth. The higher level of AAP in the foetal liver compared with that of brain is possibly sufficient to explain why cancer is not induced in liver in spite of the high rate of DNA replication. The results are consistent with the concept that replication of alkylated DNA is an essential event in initiation. There may be no quantitative relationship between replication and repair and susceptibility to cancer on comparison of different tissues, owing to the fact that, at the cellular level, cancer is a rare event. The amount of mispairing at replication necessary to bring it about may depend on the detailed organisation of the genome, and hence on cell type.Abbreviations AAP alkyl acceptor protein - NMU N-nitroso-N-methyl-urea - NEU N-Nitroso-N-ethyl-urea - O⁶MG O⁶-methylguanine - O⁶EG O⁶-ethylguanine - S-Me-Cys S-methylcysteine - PCA perchloric acid Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthday     

[35S]methionine interaction with rat liver tRNA and effect of chemical carcinogens

The interaction of [³⁵S]methionine with hepatic tRNA in normal, carcinogen-treated, and partially hepatectomized rats was studied. tRNA was preferentially labeled following [³⁵S]methionine (1.6 mCi, 25 mg/kg body wt) administration by intraperitoneal injection. The extent of [³⁵S]methionine-tRNA interaction was impaired by partial hepatectomy and by conditions having a carcinogenic potential.Presented at the Proceedings of the International Meetings on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989.Supported by CNR, Progetti Finalizzati Chimica Fine ed Oncologia     

Contamination of toiletries and cosmetic products with volatile and nonvolatile N-nitroso carcinogens

Summary Commercially available cosmetics and toiletries were analyzed for contamination with volatile and nonvolatile N-nitrosamines. Of a total of 145 samples analyzed 50 were found to contain N-nitrosodimethylamine (max. value found 24 g/kg), 26 samples were contaminated with N-nitrosomorpholine (max. value found 640 g/kg), and 25 samples contained N-nitrosodiethanolamine, a non-volatile carcinogen (max. value found 1400 g/kg). The results are discussed and compared with other published data on NDEIA in cosmetics, with reference to potential human exposure and to possible preventive measures.Abbreviations NDMA N-nitrosodimethylamine - NMOR N-nitrosomorpholine - NDEIA N-nitrosodiethanolamine Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthday     

DNA-repair methyltransferase as a molecular device for preventing mutation and cancer

Alkylation of DNA at theO ⁶ position of guanine is regarded as one of the most critical events leading to induction of mutations and cancers in organisms. OnceO ⁶-methylguanine is formed, it can pair with thymine during DNA replication, the result being a conversion of the guanine cytosine to an adenine·thymine pair in DNA, and such mutations are often found in tumors induced by alkylating agents. To counteract such effects, organisms possess a mechanism to repairO ⁶-methylguanine in DNA. An enzyme,O ⁶-methylguanine-DNA methyltransferase, is present in various organisms, from bacteria to human cells, and appears to be responsible for preventing the occurrence of such mutations. The enzyme transfers methyl groups fromO ⁶-methylguanine and other methylated moieties of the DNA to its own molecule, thereby repairing DNA lesions in a single-step reaction. To elucidate the role of methyltransferase in preventing cancers, animal models with altered levels of enzyme activity were generated. Transgenic mice carrying the foreign methyltransferase gene with functional promoters had higher levels of methyltransferase activity and showed a decreased susceptibility toN-nitroso compounds in regard to liver carcinogenesis. Mouse lines deficient in the methyltransferase gene, which were established by gene targeting, exhibited an extraordinarily high sensitivity to an alkylating carcinogen.Abbreviations MNNG N-methyl-N-nitro-N-nitrosoguanidine - MNU N-methyl-N-nitrosourea Dedicated to Dr. Haruo Sugano on the occasion of his 70th birthday. The material of this paper was essentially presented at the 60th Anniversary Symposium of the Cancer Institute and the Cancer Institute Hospital, Tokyo, held in September 1994     

O6-methylguanine repair in liver cells in vivo: Comparison between G1- and S-phase of the cell cycle

Summary To compare the formation and persistance of alkylated DNA bases in the G₁- and S-phase compartments in liver in vivo, regnerating rat liver was exposed to [¹⁴C]dimethylnitrosamine (0.57 mg/kg, IP injection) or N-[methyl ¹⁴C]-N-nitrosourea (3.3 mg/kg, intraportal injection) during the G₁ phase of the cell cyle (12 h after partial hepatectomy), or at 24 h after partial hepatectomy with 30% hepatocytes in DNA synthesis, or at 43 h after partial hepatectomy, 4 h after an hydroxyurea block from 14 to 39 h after operation with 80% hepatocytes in DNA synthesis. At 120 min after dimethylnitrosamine and 90 s, 5, 10, or 60 min after the intraportal pulse of N-methyl-N-nitrosourea the molar fractions of 7-methylguanine (7megua), O⁶-methylguanine (O⁶megua), and 3-methyladenine (3mead) and of metabolically labeled guanine were determined from DNA hydrolysates by Sephadex-G10 radiochromatography. After dimethylnitrosamine only minor differences were observed for 7megua formation in the three groups; the 3mead/7megua ratio remained constant irrespective of the number of cells in S phase. In contrast, the O⁶megua/7megua ratio revealed a loss of O⁶megua, the extent of which appeared proportional to the fraction of DNA-synthesizing cells in the liver. The rapid loss of O⁶megua in S-phase cells was confirmed after intraportal administration of N-methyl-N-nitrosourea. During the first 10 min after the methylnitrosourea pulse the O⁶megua/7megua ratio was constant in G₁ cells and dropped from 90 s to 10 min by about 15% in liver containing 30% S-phase cells and by about 40% with 80% cells in DNA synthesis. DNA-synthesizing hepatocytes are apparently endowed with a higher O⁶megua DNA transferase activity than nonproliferating liver cells. The rapid, though exhaustible elimination of O⁶megua during S-phase might result in partial protection of DNA-synthesizing cells from base-mispairing and/or from hypomethylation at G-C sites.Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdaySupported by grants from the Deutsche Forschungsgemeinschaft and the Dr. Mildred Scheel-Stiftung     

The use of radioimmunoassay to study the formation and disappearence of O6-methylguanine in mouse liver satellite and main-band DNA following dimethylnitrosamine administration

Summary A sensitive radioimmunoassay for O⁶-methyldeoxyguanosine has been developed, permitting the analysis of microgram amounts of DNA. This technique has been used in the study of the formation and removal of O⁶-methylguanine in mouse liver satellite and main-band DNA. The results indicate a reduced extent of O⁶-methylguanine formation in satellite DNA but similar rates of its removal from both classes of DNA.Abbreviations BSA bovine serum albumin - PBS phosphate-buffered saline (140 mM NaCl, 2 mM KCl, 5 mM Na₂HPO₄, 5 mMKH₂PO₄, pH 6.9)     

Interaction of the oncogene protein myc with specific DNA fragments

Summary The p110 ^gag-myc protein coded for by the retrovirus MC29 was purified 3,000-fold from MC29-Q8 transformed cells by immuno-affinity chromatography using IgG specific for the N-terminal region of the gag protein. Interaction of the protein with DNA fragments was studied by filter binding assay. DNA fragments were obtained from a MC29 DNA clone by restriction endonuclease treatment. Besides the complete DNA provirus the clone contained flanking cellular sequences into which the provirus had integrated. The DNA fragments which were retained by the p110 ^gag-myc protein were eluted from the filter and analyzed by agarose gel electrophoresis. Preferential binding of a DNA fragment originating from the flanking cellular sequences was detected. The protein did not preferentially bind to the viral LTR promoter/enhancer region as suggested by an autoregulatory model, which can therefore no longer be substantiated.Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, Februar 28–March 1, 1986     

DNA repair synthesis in fibroblast strains from patients with actinic keratosis,squamous cell carcinoma,basal cell carcinoma,or malignant melanoma after treatment with ultraviolet light,N-acetoxy-2-acetyl-aminofluorene,methyl methanesulfonate,and N-methyl-N-nitrosourea

Summary Fibroblast strains derived from skin biopsies of patients with actinic keratosis (6), malignant melanoma (18), squamous cell carcinoma (11), and basal cell carcinoma (12) were investigated for DNA repair synthesis, with 16 fibroblast strains for normal donors as controls. Cells were exposed to UV light, the UV-like carcinogen (Ac)₂ONFln, and the methylating carcinogenes MeSO₂OMe and MeNOUr. Dose-response experiments, which included 10 dose levels, were performed, the data analyzed by linear regression, and the slope of the regression line (term: G ₀) used as a measure of DNA repair synthesis. The mean experimental variability of G ₀ of individual fibroblast strains was 9.5%–15.4%, depending upon exposure. For comparison of all cell strains belonging to the same skin malignancy group with those of the control group, G ₀ values of the individual strains were combined to yield group-specific weighted mean G ₀ values.In addition, the capacity to incise UV-damaged DNA was measured in 24 cell strains from patients with skin tumors using the alkaline elution technique. For quantitating DNA-incising capacity, the initial velocities of the elution curves were plotted versus the UV dose, and the slope of the resulting regression line was used to obtain the characteristic value E ₀. The mean experimental variability of E ₀ of individual strains was ±22%. These E ₀ values were combined to yield weighted mean values of groups.The fibroblast strains in the groups of patients with actinic keratosis and malignant melanoma were found to have normal mean G ₀ values when DNA repair synthesis was challenged with UV light or one of the three carcinogens. However, the squamous cell carcinoma group exhibited significantly lower mean G ₀ values after treatment with UV light (82% that of normal donors), (Ac)₂ONFln (70%), MeSO₂OMe (70%), and MeNOUr (69%). The basal cell carcinoma group showed significantly diminished repair synthesis upon treatment with UV light (81% that of normal donors) and MeSO₂OMe (67%). In contrast to these findings, in no skin malignancy group was post UV DNA-incising capacity (E ₀) significantly diminished, although it should be noted that group sizes were only half as large as for G ₀ determinations.These data may be interpreted as indicating that DNA excision repair is impaired in fibroblast strains from patients with squamous cell carcinoma and — to a lesser extent — basal cell carcinoma. This deficiency seems to pertain to several DNA repair mechanisms, as excision of both alkylation and UV-induced damage is involved. Although the repair impairments are statistically significant, the relative risks at which the investigated patients are do not seem to be high enough as to be of immediate practical value. Our results indicate further studies would be useful.Abbreviations XP xeroderma pigmentosum - UV light ultraviolet light - UVB UV light with the wavelength from 290 nm to 320 nm - (Ac)₂ONFln N-acetoxy-2-acetylaminofluorene - MeSO₂OMe methyl methanesulfonate - MeNOUr N-methyl-N-nitrosourea - ara-C 1--d-arabinofuranosyl cytosine This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136Dedicated to Professor E. Hecker on the occasion of his 60th birthday     

Biological effects and catabolic metabolism of 3',5'-cyclic nucleotides and derivatives in rat adipose tissue and liver

Experiments on the catabolism of N⁶, O^2′-dibutyryl cyclic AMP and N⁶-monobutyryl cyclic AMP in rat liver and epididymal adipose tissue were described. During the regulation by the dibutyryl cyclic nucleotide of several parameters of the metabolism of epididymal adipose tissue adipocytes (lipolysis and glucose metabolism), neither of the butyryl groups was removed enzymatically, and nonenzymatic formation of butyric acid amounted to no more than 4 per cent over 2 hours of incubation. Therefore, intact adipocytes exhibited no deacylase activities. Furthermore, cyclic nucleotide phosphodiesterases, isolated from subcellular fractions derived from homogenates of liver and adipose tissue, which actively hydrolyzed cyclic AMP, GMP, IMP, UMP, and TMP, but not cyclic CMP, were sterically hindered by substituent groups on the N⁶ and O^2′ positions of cyclic AMP; that is, the enzyme was completely inactive against N⁶, O^2′-dibutyryl cyclic AMP and exhibited only a trace amount of activity against N⁶-monobutyryl cyclic AMP. However, mechanisms do exist in these tissues for the removal of one or both butyryl groups; subcellular fractions derived from tissue homogenates contained an N-acyl hydrolase active against N⁶, O^2′-dibutyryl and N⁶-monobutyrl cyclic AMP, and, possibly, also esterase activity against the former. The relationships of function (as lipolytic agents and substrates for cyclic nucleotide phosphodiesterases) to structure among a variety of 3′, 5′-cyclic nucleotides were investigated using adipocytes and partially purified enzymes. An incubation medium consisting of a sodium chloride-phosphate buffer devoid of Mg²⁺, Ca²⁺, and K⁺ permitted entry of all nucleotides into adipocytes. In such permeable cells, all purine and pyrimidine cyclic nucleotides, with the exception of the thymine analogs, stimulated lipolytic activity to various extents; however, no relationships, inverse or otherwise, existed between the potencies of the compounds as activators of lipase activity and their susceptibility to hydrolysis by the cyclic nucleotide phosphodiesterase. Combinations of saturating concentrations of cyclic nucleotides produced rates of lipolysis in cells that were similar to the rate produced by the more potent member of each pair when tested alone; only the lipolytically inactive thymine cyclic nucleotides inhibited the effects of other cyclic nucleotides upon lipolysis. Such results indicated that all active cyclic nucleotides stimulated the same lipolytic mechanism in adipose tissue.     

Proinsulin synthesis in islets of Langerhans from spiny mice (Acomys cahirinus). comparison with rats and mice

Summary Proinsulin synthesis in response to different concentrations of glucose has been studied in islets of Langerhans of acomys, rat and mouse. The response to increased glucose was small in acomys when compared with rat or mouse. The time course of the effect of glucose on proinsulin synthesis was also studied in sequential 15 min periods up to 60 min. With 5.5 mM glucose the rate of³H-leucine incorporation for acomys islets was greater over the periods 15–30, 30–45 and 45–60 min after the addition of glucose to the islets than at 0–15 min. By contrast rat islets exhibited a greater glucose-induced stimulation between 15–30 and 30–45 min relative to the 0–15 min period and a further increase of the incorporation rate between 45 and 60 min. With 27.5 mM glucose acceleration of incorporation relative to the first 15 min period occurred at 15–30 min; it was then stable up to 60 min for islets both from acomys and from rat. Incorporation into total protein was much greater in acomys than in rat or mouse: thus in 2.75 mM glucose, acomys islets incorporated 5 and 19 times more³H-leucine into total protein than did islets from rat or mouse respectively. In 27.5 mM glucose the incorporation into total protein of the islets from acomys was 4 and 13 times greater. Islet content of insulin was similar for all three species. No significant changes in ATP content were observed in response to changes in glucose concentration from 2.75 to 27.5 mM. The results demonstrate a decreased responsiveness of proinsulin synthesis to glucose in acomys and are discussed in terms of the known decreased sensitivity of insulin release in this animal.This work was supported by Grant No. 3106073 from the Fonds National Suisse pour la Recherche Scientifique     

Global gene expression analysis in liver of obese diabetic db/db mice treated with metformin

Aims/hypothesis Metformin is widely used as a hypoglycaemic reagent for type 2 diabetes. While the reduction of hepatic gluconeogenesis is thought to be a key effect, the detailed molecular mechanism of action of metformin remains to be elucidated. To gain insight into this, we performed a global gene expression profiling study.Materials and methods We performed DNA microarray analysis to study global gene expression in the livers of obese diabetic db/db mice 2 h after a single administration of metformin (400 mg/kg).Results This analysis identified 14 genes that showed at least a 1.5-fold difference in expression following metformin treatment, including a reduction of glucose-6-phosphatase gene expression. The mRNA levels of glucose-6-phosphatase showed one of the best correlations with blood glucose levels among 12,000 genes. Enzymatic activity of glucose-6-phosphatase was also reduced in metformin-treated liver. Moreover, intensive analysis of the expression profile revealed that metformin effected significant alterations in gene expression across at least ten metabolic pathways, including those involved in glycolysis-gluconeogenesis, fatty acid metabolism and amino acid metabolism.Conclusions/interpretation These results suggest that reduction of glucose-6-phosphatase activity, as well as suppression of mRNA expression levels of this gene, in liver is of prime importance for controlling blood glucose levels in vivo, at least at early time points after metformin treatment. Our results also suggest that metformin not only affects expression of specific genes, but also alters the expression level of multiple genes linked to the metabolic pathways involved in glucose and lipid metabolism in the liver.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Xeroderma pigmentosum patients from the federal Republic of Germany: Decrease in post-UV colony-forming ability in 30 xeroderma pigmentosum fibroblast strains is quantitatively correlated with a decrease in DNA-incising capacity

Summary A total of 16 normal and 46 XP fibroblast strains from the Mannheim Collection were investigated for colony-forming ability following exposure to both UV light and the UV-like carcinogen (Ac)₂ONFln. The dose-response experiments included up to 13 dose levels. The exponential segments of the curves were analysed by linear regression and the negative reciprocal of the regression coefficient (D₀) was calculated for each cell strain.For quantitating the DNA-incising capacity, DNA elution curves were determined at several UV dose levels. Plotting the initial velocities of the elution curves versus the UV dose yielded a regression line, the slope of which was used to obtain the characteristic value E₀.Comparing D₀ with E₀ values showed that cell strains in which colony-forming ability was reduced suffered a reduction of DNA-incising capacity of the same magnitude. There were only 3 exceptional strains in which reduction of DNA-incising capacity was less pronounced than reduction of colony-forming ability. We have previouly shown (Fischer et al. 1982) that D₀ values from 27 XP strains of the Mannheim Collection were correlated with clinical symptoms. This correlation is now being extended by relating colony-forming ability to the magnitude of the DNA incision defect. From our data we conclude that the best quantitative biochemical denominator to explain the sun sensitivity of XP is that of a defective incision of UV-damaged DNA.A considerable similarity in sensitivity towards both UV light and (Ac)₂ONFln was found in 16 normal and 46 XP strains. This seems to indicate that UV-and (Ac)₂ONFln-induced DNA damage are removed to a large extent by the same pathways in human fibroblasts.Abbreviations XP xeroderma pigmentosum - (Ac)₂ONFln N-acetoxy-2-acetylaminofluorene - UV light ultraviolet light - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - ara-C 1--d-arabinofuranosyl cytosine This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

Effect of substrate interactions on in vitro fatty acid synthesis in adipose tissue of normal and genetically obese mice

Summary Fatty acid synthesis was measured in vitro in pieces of adipose tissue from lean and obese-hyperglycaemic (ob/ob) mice, using ¹⁴C-glucose or ¹⁴C-lactate and ³H₂O to obtain absolute rates of total fatty acid synthesis. In the presence of lipoproteintriglyceride (2.5 mol/l) metabolic interaction occurred which decreased glucose incorporation into fatty acids by 30% in lean mouse tissue, but not in obese mouse tissue. In the absence of added insulin, the contribution of glucose to total fatty acid synthesis was 69% in obese mouse tissue, significantly lower than the value of 87% obtained in lean mouse tissue. Insulin increased the contribution of glucose to total synthesis in both lean and obese mouse tissues, although the value in obese mouse tissue (83%) remained lower than the value in lean mouse tissue (100%). Lactate was not a major precursor for fatty acid synthesis. When both lactate (2 mmol/l) and glucose (15 mmol/l) were present, the contribution of lactate to total fatty acid synthesis was not increased in obese mouse tissue, suggesting that even in the presence of insulin, about 30% of the carbon was provided by intracellular precursors.     

Intragastric nicotine protects against 40% ethanol-induced gastric mucosal injury despite pretreatment with propranolol orN-ethylmaleimide in rats

We tested the hypotheses that the protective effect of intragastric nicotine against ethanol-induced gastric mucosal injury is dependent on propranolol- orN-ethylmaleimide-sensitive mechanisms. Propranolol was administered in doses (2 and 20 mg/kg) that provided dose-related blockade of -adrenoceptors (significant decreases in heart rate).N-Ethylmaleimide was administered in doses that previously had been shown to increase gastric vascular permeability (10 mg/kg) or inhibit gastric mucosal sulfhydryl compounds (50 mg/kg). At 0.5 hr after these or control subcutaneous pretreatments, the rats received intragastric nicotine (4 mg/kg) or vehicle. One hour later 40% ethanol was given intragastrically. The gastric corpus mucosal lesions were recorded by polaroid photographs after another hour, and their areas measured unbiasedly by computerized image analysis. The results showed thatN-ethylmaleimide, but not propranolol, aggravated ethanol-induced gastric mucosal injury. The protective effect of intragastric nicotine was not modified by either pretreatment. We conclude that the mechanism mediating intragastric nicotine protection against 40% ethanol-induced gastric mucosal injury is independent of propranolol- orN-ethylmaleimide-sensitive mechanisms.Supported by Veterans Administration Medical Research Funds, and in part by research grants (0162-01, 02 and 0291-01) from the Smokeless Tobacco Research Council, Inc., and by funds (1RT 80) provided by the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco-Related Disease Research Program of the University of California to FWL. Dr. Endoh is a recipient of the University of California Tobacco-Related Disease Research Program Research Fellowship Award (FT 37).     

Control of cardiac myofilament activation and PKC-betaII signaling through the actin capping protein, CapZ

Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilaments from transgenic mice (TG-CapZ) expressing a reduced amount of CapZ demonstrate altered function and protein kinase C (PKC) signaling [Pyle WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, and Solaro RJ., Circ. Res. 90 (2002) 1299-306]. The aims of the current study were to determine the direct effects of CapZ on myofilament function and on PKC signaling to the myofilaments. Our studies compared mechanical properties of single myocytes from TG-CapZ mouse hearts to wild-type myocytes from which CapZ was extracted using PIP(2). We found that myofilaments from CapZ-deficient transgenic myocardium exhibited increased Ca(2+) sensitivity and maximum isometric tension. The extraction of CapZ from wild-type myofilaments replicated the increase in maximum isometric tension, but had no effect on myofilament Ca(2+) sensitivity. Immunoblot analysis revealed that the extraction of CapZ was associated with a reduction in myofilament-associated PKC-beta(II) and that CapZ-deficient transgenic myofilaments also lacked PKC-beta(II). Treatment of wild-type myofilaments with recombinant PKC-beta(II) reduced myofilament Ca(2+) sensitivity, whereas this effect was attenuated in myofilaments from TG-CapZ mice. Our results indicate that cardiac CapZ directly controls maximum isometric tension generation, and establish CapZ as an important component in anchoring PKC-beta(II) at the myofilaments, and for mediating the effects of PKC-beta(II) on myofilament function.     

cAMP inhibition of murine intestinal Na/H exchange requires CFTR-mediated cell shrinkage of villus epithelium

BACKGROUND AND AIMS: Unlike the intestine of normal subjects, small-intestinal epithelia of cystic fibrosis patients and cystic fibrosis transmembrane conductance regulator protein-null (CFTR(-)) mice do not respond to stimulation of intracellular cyclic adenosine monophosphate with inhibition of electroneutral NaCl absorption. Because CFTR-mediated anion secretion has been associated with changes in crypt cell volume, we hypothesized that CFTR-mediated cell volume reduction in villus epithelium is required for intracellular cyclic adenosine monophosphate inhibition of Na(+)/H(+) exchanger (primarily Na(+)/H(+) exchanger 3) activity in the proximal small intestine. METHODS: Transepithelial (22)Na flux across the jejuna of CFTR(+), CFTR(-), the basolateral membrane Na(+)/K(+)/2Cl(-) co-transporter protein NKCC1(+), and NKCC1(-) mice were correlated with changes in epithelial cell volume of the midvillus region. RESULTS: Stimulation of intracellular cyclic adenosine monophosphate resulted in cessation of Na(+)/H(+) exchanger-mediated Na(+) absorption (J(ms)(NHE)) in CFTR(+) jejunum but had no effect on J(ms)(NHE) across CFTR(-) jejunum. Cell volume indices indicated an approximately 30% volume reduction of villus epithelial cells in CFTR(+) jejunum but no changes in CFTR(-) epithelium after intracellular cyclic adenosine monophosphate stimulation. In contrast, cell shrinkage induced by hypertonic medium inhibited J(ms)(NHE) in both CFTR(+) and CFTR(-) mice. Bumetanide treatment to inhibit Cl(-) secretion by blockade of the Na(+)/K(+)/2Cl(-) co-transporter, NKCC1, of stimulated CFTR(+) jejunum prevented maximal volume reduction of villus epithelium and recovered approximately 40% of J(ms)(NHE). Likewise, J(ms)(NHE) and cell volume were unaffected by intracellular cyclic adenosine monophosphate stimulation in NKCC1(-) jejuna. CONCLUSIONS: These findings show a previously unrecognized role of functional CFTR expressed in villus epithelium: regulation of Na(+)/H(+) exchanger 3-mediated Na(+) absorption by alteration of epithelial cell volume.     

Alterations in mitochondrial function as a harbinger of cardiomyopathy: Lessons from the dystrophic heart

While compelling evidence supports the central role of mitochondrial dysfunction in the pathogenesis of heart failure, there is comparatively less information available on mitochondrial alterations that occur prior to failure. Building on our recent work with the dystrophin-deficient mdx mouse heart, this review focuses on how early changes in mitochondrial functional phenotype occur prior to overt cardiomyopathy and may be a determinant for the development of adverse cardiac remodelling leading to failure. These include alterations in energy substrate utilization and signalling of cell death through increased permeability of mitochondrial membranes, which may result from abnormal calcium handling, and production of reactive oxygen species. Furthermore, we will discuss evidence supporting the notion that these alterations in the dystrophin-deficient heart may represent an early “subclinical” signature of a defective nitric oxide/cGMP signalling pathway, as well as the potential benefit of mitochondria-targeted therapies. While the mdx mouse is an animal model of Duchenne muscular dystrophy (DMD), changes in the structural integrity of dystrophin, the mutated cytoskeletal protein responsible for DMD, have also recently been implicated as a common mechanism for contractile dysfunction in heart failure. In fact, altogether our findings support a critical role for dystrophin in maintaining optimal coupling between metabolism and contraction in the heart.     

Fatty acid ethyl esters cause pancreatic calcium toxicity via inositol trisphosphate receptors and loss of ATP synthesis

BACKGROUND & AIMS: Fatty acid ethyl esters are ethanol metabolites inducing sustained, toxic elevations of the acinar cytosolic free calcium ion concentration ([Ca(2+)](C)) implicated in pancreatitis. We sought to define the mechanisms of this elevation. METHODS: Isolated mouse pancreatic acinar cells were loaded with fluorescent dyes for confocal microscopy to measure [Ca(2+)](C) (Fluo 4, Fura Red), endoplasmic reticulum calcium ion concentration ([Ca(2+)](ER), Mg Fluo 4), mitochondrial membrane potential (TMRM), ADP:ATP ratio (Mg Green), and NADH autofluorescence in response to palmitoleic acid ethyl ester and palmitoleic acid (10-100 micromol/L). Whole-cell patch clamp was used to measure the calcium-activated chloride current and apply ethanol metabolites and/or ATP intracellularly. RESULTS: Intracellular delivery of ester induced oscillatory increases of [Ca(2+)](C) and calcium-activated currents, inhibited acutely by caffeine (20 mmol/L), but not atropine, indicating involvement of inositol trisphosphate receptor channels. The stronger effect of extracellular ester or acid caused depletion of [Ca(2+)](ER), not prevented by caffeine, but associated with depleted ATP, depleted NADH autofluorescence, and depolarized mitochondria, suggesting calcium-ATPase pump failure because of lack of ATP. Intracellular ATP abolished the sustained rise in [Ca(2+)](C), although oscillatory signals persisted that were prevented by caffeine. Inhibition of ester hydrolysis markedly reduced its calcium-releasing effect and consequent toxicity. CONCLUSIONS: Fatty acid ethyl ester increases [Ca(2+)](C) through inositol trisphosphate receptors and, following hydrolysis, through calcium-ATPase pump failure from impaired mitochondrial ATP production. Lowering cellular fatty acid substrate concentrations may reduce cell injury in pancreatitis.     

Efflux of radioactive nucleotides from mouse pancreatic islets prelabelled with 2-3H-adenosine

Summary Cultured mouse pancreatic islets were prelabelled with 2-³H-adenosine in order to monitor the efflux pattern of radioactivity and insulin. The outflow of radioactivity decreased continuously when the islets were perifused with glucose (1.67 mmol/1). When raising the glucose concentration to 16.7 mmol/1, there was a prompt inhibition of the radioactive efflux concomitant with an increased rate of insulin release. These effects were reversed when the high glucose challenge was withdrawn. Similar radioactive efflux patterns were obtained after addition of -ketoisocaproic acid, leucine or pyruvate to the perifusion medium, and also when the islets were challenged with high glucose concentrations in the absence of calcium. Both antimycin A and glipizide stimulated the efflux of radioactivity, although only the addition of glipizide was accompanied by a stimulation of the insulin release. Nucleotides constituted approximately 90% of the total effluent radioactivity. Decrease in the radioactive AMP and ADP efflux due to high glucose was furthermore found to be the cause of the observed inhibition of the total radioactive efflux. The changes in radioactive efflux induced by glucose probably reflect changes in the intracellular concentrations of AMP and ADP. It is concluded that no simple correlation exists between radioactive efflux and insulin release and that changes in the intracellular concentrations of nucleotides may be an early event in the stimulus-secretion coupling of glucose-induced insulin release.