Acute exposure to the nutritional toxin alcohol reduces brain protein synthesis in vivo.

Few studies have measured brain protein synthesis in vivo using reliable methods that consider the precursor pool, and there is a paucity of data on the regional sensitivity of this organ to nutritional or toxic substances. We hypothesized that different areas of the brain will exhibit variations in protein synthesis rates, which might also be expected to show different sensitivities to the nutritional toxin, ethanol. To test this, we dosed male Wistar rats with ethanol (75 mmol/kg body weight) and measured rates of protein synthesis (ie, the fractional rate of protein synthesis, defined as the percentage of the protein pool renewed each day; k(s), %/d) in different brain regions 2.5 hours later with the flooding dose method using L-[4-(3)H] phenylalanine. In the event that some regions were refractory to the deleterious effects of ethanol, we also predosed rats with cyanamide, an aldehyde dehydrogenase inhibitor (ie, cyanamide + ethanol), to increase endogenous acetaldehyde, a potent neurotoxic agent. The results indicated the mean fractional rates of protein synthesis in the cortex was 21.1%/d, which was significantly lower than either brain stem (30.2%/d, P <.025), cerebellum (30.1%/d, P <.01), or midbrain (29.8%, P <.025). Ethanol significantly decreased protein synthesis in the cortex (21%, P < 0.01), cerebellum (19%, P <.025), brain stem (44%, P <.025), but not in the midbrain (not significant [NS]). However, significant reductions in protein synthesis in the midbrain occurred in cyanamide + ethanol-dosed rats (60%, P <.0001). Cyanamide + ethanol treatment also reduced k(s) in the brain stem (66%, P <.001), cortex (59%, P <.001), and cerebellum (55%, P <.001). In conclusion, the applicability of the flooding dose technique to measure protein synthesis in the brain in vivo is demonstrated by its ability to measure regional difference. Impaired protein synthesis rates may contribute to or reflect the pathogenesis of alcohol-induced brain damage.

Functional and compositional studies of arteries stored in University of Wisconsin solution compared with Krebs-Bülbring buffer.

Endothelium-dependent and -independent vasorelaxation in ring segments of rabbit thoracic aorta is reduced and noradrenaline-induced vasoconstriction unaltered after prolonged storage in University of Wisconsin solution (UW) compared to arteries stored in extracellular-type solutions such as Krebs-Bülbring buffer (KBB). The aims of the present study were to determine whether angiotensin-II-induced vasoconstriction, alterations in myosin light chains, protein synthetic capacity, and subcellular structures are altered after 8 days of UW storage at 4 degrees C. The present study showed reduced contractility to angiotensin II, following 8 days of cold storage in UW, that was not reversed in the presence of a nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester (100 microM). Measurements of contractile protein ratios in the same tissues after cold storage in UW or KBB did not show any significant alterations in smooth muscle myosin light chains or protein synthetic capacity (reflected by total RNA). It is concluded that reductions in vasoconstriction in UW-stored tissue are unlikely to be due to increased release of nitric oxide nor reduced availability of myosin light chains for phosphorylation and vasoconstriction.

Skeletal muscle protein loss due to D-penicillamine results from reduced protein synthesis.

Reports in the literature indicate that the trifunctional amino acid D-penicillamine (D-P) induces a variety of muscle abnormalities, although the mechanisms are unknown. We hypothesised that defects may also arise due to the effects of D-P on rates of protein synthesis, possibly via changes in muscle metal composition. Male Wistar rats were injected with D-P at doses of 50 and 500 mg/kg body weight, i.p. Rats designated as controls were injected with 0.15 mol/l NaCl. After 24 h, there were reductions in muscle protein contents, protein synthetic capacities (RNA:protein ratio), fractional rates of protein synthesis, synthesis rates per unit RNA and synthesis rates per unit DNA in skeletal muscles of D-P treated rats. There were no statistically significant differences between the responses of the muscles containing a predominance of either Type I (represented by the soleus) or Type II (represented by the plantaris) fibres. In general, intracellular amino acids were not significantly affected by D-P treatment. Changes in muscle metals included significant reductions in copper, iron and manganese, without alterations in zinc or magnesium. In liver D-P reduced copper and iron though zinc, manganese and magnesium were unaffected. These effects of D-P on muscle may have been direct, as plasma indices of liver (activities of alkaline phosphatase and alanine aminotransferase) and kidney (urea, creatinine and electrolytes) damage were not significantly altered by D-P treatment. Plasma levels of corticosterone, insulin and free T3 were also not significantly affected by D-P treatment. Muscle protein carbonyl concentrations, an index of free radical activity, were similarly unaffected. This is the first report of reduced rates of muscle protein synthesis in D-P treatment. Our data suggests that the reduced rates of muscle protein synthesis may contribute to, or reflect, the muscle abnormalities observed in patients undergoing D-P treatment.

Diarrhea reduces the rates of cardiac protein synthesis in myofibrillar protein fractions in rats in vivo.

Although chronic diarrhea affects heart function and morphology, the pathogenic mechanisms are unknown. It was our hypothesis that diarrhea imposes metabolic stress to inhibit the synthesis of new contractile proteins. To test this hypothesis, we investigated the effects of lactose-induced diarrhea in rats. The groups were: 1) freely fed controls, 2) rats with lactose-induced diarrhea or 3) pair-fed rats. After 1 wk, hearts from the rats were subjected to subcellular fractionation techniques to isolate the major protein fractions, including myofibrillar proteins. The rates of protein synthesis were measured with concomitant assay of cardiac composition and plasma analytes. In comparison with the control group, diarrhea induced the following changes (P < 0.05): a decrease in heart weight, reduced RNA and mixed protein contents and a reduction in the fractional rate of mixed protein synthesis. There was a reduction in the content of all protein fractions. The fractional synthesis rate was reduced only for the myofibrillar fraction. Plasma insulin-like growth factor-I, but not corticosterone, was reduced. Plasma cholesterol and triglyceride concentrations were also reduced. In comparison with the pair-fed group, diarrhea induced the following changes (P < 0.05): a reduction in heart weight and fractional rate of mixed protein synthesis, reduced myofibrillar absolute synthesis rate and increased sarcoplasmic/myofibrillar fractional synthesis rate ratio. Plasma bicarbonate, triglyceride and urea concentrations were reduced, with an increase in albumin. Diarrhea impaired cardiac biochemistry, including a reduction in protein content and synthesis. A substantial proportion of these changes is due to anorexia, but the selective reduction in the synthesis of contractile proteins is a feature exclusive to the diarrhea group and may be due to reductions in plasma insulin-like growth factor-I.

Acute effect of ethanol on 7-hydroperoxycholesterol in muscle and liver.

We tested the hypotheses that ethanol sensitivities of muscle and liver can be discerned in the initial periods of ethanol exposure, especially when acetaldehyde levels are markedly raised with cyanamide, an aldehyde dehydrogenase inhibitor. To test this, we measured cholesterol hydroperoxides in soleus (Type I) and plantaris (Type II) muscle in four groups of rats acutely (i.e., 2.5 h) exposed to: [S] saline (control), [Cy] cyanamide, [EtOH] ethanol, or [Cy + EtOH] cyanamide + ethanol. Comparative reference was also made to the response of the liver. After 2.5 h, ethanol alone significantly increased 7 alpha-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH) and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 beta-OOH) levels in plantaris muscle. Identical qualitative effects were seen in rats treated with cyanamide + ethanol, but there was no discernible difference between groups [EtOH] and [Cy + EtOH]. In both the soleus muscle and liver, none of the treatments with either ethanol or cyanamide + ethanol had any effect on any of the measured parameters. This is the first report of a differential response of 7 alpha-OOH and 7 beta-OOH in Type II, compared to Type I predominant muscles, and the first time that muscle has been shown to be more sensitive than the liver in terms of its lipid marker response to oxidative stress. Perturbations in the muscle membrane lipid domain may contribute to impairment of muscle in alcoholism.

Contractile protein synthesis rates in vivo in the rat jejunum: modulating role of adrenalectomy and thyroidectomy on ethanol-induced changes.

Acute ethanol toxicity has many deleterious effects on the gastrointestinal tract and, in particular, inhibits small intestinal protein synthesis. The mechanism(s) of the ethanol-induced inhibition of protein synthesis are unknown. This study was designed to investigate the role of adrenal and thyroid hormones on the ethanol-induced inhibition of whole jejunal protein synthesis in adrenalectomised and thyroidectomised male Wistar rats. Acute ethanol dosage significantly reduced protein synthesis in all subcellular protein fractions of the whole jejunum, in sham-thyroidectomized, thyroidectomized, sham-ardrenalectomized and adrenalectomized rats. Synthesis rates relative to RNA and DNA were also highly significantly reduced in all treatment groups in ethanol-dosed rats. However in most protein fractions the inhibition of protein synthesis was greater in thyroidectomized compared to sham-thyroidectomized rats, whereas the reverse was true in adrenalectomized rats. Neither adrenalectomy or thyroidectomy completely abolished ethanol-induced inhibition in protein synthesis, suggesting that ethanol or its metabolites, e.g. acetaldehyde, acts directly on the tissue. However, the ethanol-induced inhibition of protein synthesis was greater in thyroidectomized rats demonstrating that contractile and non-contractile proteins synthesis in the jejunum is under control of complex regulatory processes.

Brain nucleic acid composition and fractional rates of protein synthesis in response to chronic ethanol feeding: comparison with skeletal muscle.

Brain atrophy is a common feature of chronic alcohol misuse, although the pathogenic mechanisms are unknown. We propose that defects in protein synthesis are contributing events. To test this hypothesis the experimental effects of chronic (i.e., 2 and 3 weeks) ethanol feeding on brain nucleic acid composition and rates of protein synthesis in vivo were investigated. These were compared with those of skeletal muscle (represented by the plantaris). Male Wistar rats, used at mean body weights of either 82 g (first study for 2 weeks ) or 93 g (second study for 3 weeks) were fed a nutritionally complete liquid diet in which ethanol comprised a third of the total calories. Control rats were pair-fed identical amounts of the same diet, in which ethanol was substituted by isoenergetic glucose. At 2 weeks there were small reductions (i.e., approximately 5-10%) in the weight of the whole brain, cortex, and brain stem. Ethanol-induced reductions in the total protein content of the brain stem was found at 2 weeks, although these changes did not achieve significance. At 3 weeks the weights of whole brain were significantly reduced compared to a greater reduction in skeletal muscle weights. Total protein contents were reduced at 3 weeks in the whole brain and skeletal muscle. At 2 weeks there were decreases in the RNA contents of the cortex, brain stem, and entire brain. There were also reductions in cerebellum RNA composition only when expressed relative to DNA. The DNA composition of the brain was relatively unaffected by chronic ethanol feeding. At 3 weeks, total RNA and DNA were reduced in the whole brain and muscle. Fractional rates of protein synthesis (i.e., the percentage of tissue protein pool renewed each day) in the brain were unaltered after 3 weeks of ethanol feeding, but were reduced in skeletal muscles, largely as a consequence of reduced RNA composition. In conclusion, only moderate changes in the brain were found in ethanol feeding. These data can be compared to skeletal muscle, which shows that ethanol induces profound reductions in protein, RNA, and protein synthesis rates.

Application of proton NMR spectroscopy to measurement of whole-body RNA degradation rates: effects of surgical stress in human patients.

The urinary catabolites, N2,N2-dimethylguanosine (DMG), pseudouridine (PSU) and 7-methylguanine (m7-Gua) are formed from post-transcriptional methylation of RNA bases and are not reincorporated into RNA upon its degradation. Their quantitative urinary excretion may be used to determine rates of whole body degradation of individual RNA species since DMG occurs exclusively in tRNA, PSU occurs in rRNA and tRNA and m7-Gua occurs in all RNA species. Conventional HPLC analysis has several drawbacks since pre-analytical steps may involve selective losses and, under certain conditions, other urinary analytes may co-elute. In the present paper, we report analysis of these compounds by high-field 1H-nuclear magnetic resonance (1H-NMR) spectroscopy. Urinary concentrations of these metabolites were found to be in agreement with previously published HPLC and ELISA determinations. However, NMR analysis required minimal sample preparation (other than lyophilisation and reconstitution) and was capable of the simultaneous determination of other relevant analytes such as creatinine. This technique was therefore applied to urine samples from patients who had undergone surgical stress and insulin-like growth factor-1 (IGF-I) therapy. Surgical stress increased the excretion of DMG and m7-Gua. Degradation rates for tRNA and mRNA were also higher in surgically stressed subjects when compared with controls but degradation rates of rRNA decreased by approx. 30%. However, injection of IGF-I (40 micrograms/kg s.c.) had no significant effect on the excretion of these nucleosides. These data indicated that IGF-I therapy has no marked effects on RNA turnover following trauma. We suggest that this technique can be applied to study of RNA metabolism in any surgical or medical condition. Furthermore, since only 0.6 ml of urine is required, studies in neonates seem to be feasible.

Osmotic diarrhoea and skeletal muscle protein synthesis in vivo.

The pathogenic nature of the wasting seen in diarrhoea is unknown. This study measured protein synthesis in an established model of diarrhoea using lactose for seven days. Comparisons were also made with data obtained from rats fed an identical diet in which lactose was replaced by isocaloric glucose ad libitum (that is, the control diet). To account for diarrhoea induced anorexia, a third group of rats were included, which were fed identical amounts of the control diet as the rats with diarrhoea inducing diet. Comparisons of the diarrhoea induced group with rats fed the control diet ad libitum showed that diarrhoea caused a significant reduction in body weights. Type I and type II muscles showed significant reductions in protein, RNA, and DNA contents, as well as a fall in the derived parameters, RNA/DNA, protein/DNA, and RNA/protein. Fractional rates of protein synthesis (ks) were also reduced. However, synthesis rates of type I and II muscles relative to RNA (kRNA) were unchanged in these muscles in diarrhoea induced rats compared with ad libitum fed controls. In the jejunum there was an increase in the RNA/DNA ratio, and reductions in ks and kRNA. Comparisons were also made between rats with diarrhoea and rats pair fed the control diet. There were no changes in total muscle protein, RNA or DNA contents. This suggests that an important feature of body wasting in diarrhoea is the element of anorexia, which induces severe metabolic changes. The comparison between rats with diarrhoea and the pair fed group showed that histological features of the plantaris were not overtly changed, though diarrhoea caused significant reductions in RNA/DNA, protein/DNA, ks, and kRNA. Similar changes were seen for the soleus; though the reduction in ks failed to attain statistical significance. In the jejunum a comparison of diarrhoea induced rats with pair fed controls, showed increases in the ratios of RNA/DNA and protein/DNA.

The effects of acute administration of ethanol on jejunal protein synthesis and circulating insulin-like growth factor (IGF)-1 and IGF binding proteins in ad libitum fed and nutritionally restricted rats.

The effects of acute ethanol administration (75 mmol/kg body weight) to male Wistar rats (either ad libitum fed or nutritionally restricted) on fractional rates of protein synthesis in the jejunum was assessed together with the changes in IGF-1 and IGF binding protein concentrations. Acute administration of ethanol resulted in significant decreases in fractional rates of protein synthesis in the whole jejunum and jejunal seromuscular layers of both the ad libitum fed and nutritionally restricted animals. The synthesis rate per unit RNA (k(RNA), mg protein/day/mg RNA) in whole jejunum was reduced by 29% and 24% in the nutritionally restricted and ad libitum fed animals, respectively. Mean IGF-1 levels were lower in the nutritionally restricted group (871 +/- 36.9 microg/l) than the ad libitum animals (960 +/- 27.3 microg/l) although this did not reach significance. In contrast, administration of alcohol to both groups markedly reduced circulating IGF-1 levels (ad libitum: 518 +/- 19.8 microg/l, nutritionally restricted: 417 +/- 33.7 microg/l). Furthermore, ethanol treatment resulted in a three-fold increase in the intensity of a 30 kDa IGF binding protein (IGFBP) in the ad libitum fed animals and a fourfold increase in both 30 and 32 kDa IGFBP bands in the nutritionally restricted group as visualized by Western ligand blotting. Decreases in levels of IGF-1 allied with increased circulating small molecular weight IGFBPs may contribute to the reduction in fractional rates of protein synthesis in the gastrointestinal tract of ethanol-treated rats.

Characterization of contractile and non-contractile protein synthesis in the stomach, small and large intestine and caecum of the rat, and response to acute ethanol dosage.

An investigation was made into the relative composition and synthesis rates of smooth muscle contractile proteins in vivo in different regions of the rat gastrointestinal tract. There was considerable homogeneity in the composition of sarcoplasmic proteins in the small bowel (i.e. 54-58 mg/g) but considerable variability in the large bowel, i.e. highest in the caecum (97 mg/g) and lowest in the colon (21 mg/g). The myofibrillary protein concentration was constant throughout the gastrointestinal tract, i.e. 20-34 mg/g. Stromal fractions varied from 6 to 39 mg/g and was highest in the cardiac region of the stomach and lowest in the duodenum. Fractional rates of protein synthesis were measured with a flooding dose of L-[4-(3)H]phenylalanine. In control rats synthesis rates in sarcoplasmic protein fractions were relatively much higher (43-107%/day) than myofibrillar (27-52%/day) or stromal (6-26%/day) proteins. Fractional rates of stromal protein synthesis showed the greatest variability while myofibrillar synthesis rates the least, throughout the alimentary tract. Jejunal seromuscular layer myofibrillar proteins had the highest synthesis rates (49%/day). In response to acute ethanol injection, protein synthesis in all jejunal fractions fell by 20-30%. Contractile and non-contractile proteins from the cardiac region of the stomach, duodenal seromuscular layer and large bowel seromuscular layer were insensitive to ethanol administration. Protein synthesis of sarcoplasmic proteins from the antrum, ileum seromuscular layer and myofibrillar proteins from the ileum seromuscular layer and caecum were also significantly depressed as a result of ethanol treatment.

Lysosomal and nonlysosomal protease activities of the brain in response to ethanol feeding.

The contribution of impaired degradative processes to the cellular changes occurring in the brain as a consequence of chronic ethanol exposure was assessed. Male Wistar rats were fed nutritionally adequate liquid diets containing ethanol as 35% of total dietary calories. Controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. The results showed that at the end of 3 weeks the activities of neutral protease (nonlysosomal) and cathepsin D (lysosomal) were unaltered. However, there were significant elevations in the activities of the lysosomal enzyme cathepsin B, regardless of whether the activities were expressed relative to wet weight ( p = 0.005), protein (p = 0.006), or DNA (p = 0.045). In addition, we showed that the activities of cathepsin B were not significantly affected by additions of carnosine or acetaldehyde, in vitro. However, neutral protease activities were increased by carnosine additions in vitro. We conclude that selective alterations in brain protease activities may be contributing factors in the genesis of alcoholic brain disorders.

The acute effects of ethanol and acetaldehyde on the synthesis of mixed and contractile proteins of the jejunum.

An investigation was made into the acute effects of ethanol and acetaldehyde with or without enzyme inhibitors of alcohol dehydrogenase (4-methylpyrazole) and aldehyde dehydrogenase (cyanamide) on fractional rates of protein synthesis of mixed and contractile proteins of the jejunum. Ethanol decreased the fractional rates of mixed and contractile protein synthesis (i.e. ks, defined as the percentage of tissue protein renewed each day) by approximately 25%. Pretreatment with 4-methylpyrazole followed by treatment with ethanol further reduced mixed and contractile ks by approximately 30%, when compared with saline plus saline and 4-methylpyrazole plus saline groups. The greatest reductions in ks of mixed and contractile proteins occurred with cyanamide pretreatment followed by ethanol treatment; mixed and contractile protein ks in the cyanamide plus ethanol group decreased by approximately 60% when compared with saline plus saline and cyanamide plus saline groups, whereas ks decreased by approximately 45% when compared with the saline plus ethanol injected group. Acetaldehyde treatment alone caused no significant inhibition of protein synthesis. However, 4-methylpyrazole pretreatment plus acetaldehyde treatment significantly reduced mixed and contractile ks by approximately 20% when compared with the saline group, and by approximately 15% when compared with the 4-methylpyrazole plus saline and saline plus acetaldehyde groups. These data show that ethanol alone and perhaps high levels of acetaldehyde may be responsible for the inhibition of intestinal protein synthesis and related pathological derangements, e.g. motility disturbances due to loss of contractile proteins.