Glutamine metabolism in sepsis and infection.

Severe infection causes marked derangements in the flow of glutamine among organs, and these changes are accompanied by significant alterations in regional cell membrane transport and intracellular glutamine metabolism. Skeletal muscle, the major repository of glutamine, exhibits a twofold increase in glutamine release during infection, which is associated with a significant increase in endogenous glutamine biosynthesis. Despite an increase in glutamine synthetase activity in skeletal muscle, the intracellular glutamine pool becomes depleted, indicating that release rates exceed rates of synthesis. Simultaneously, the circulating pool of glutamine does not increase, indicating accelerated uptake by other organs. The liver appears to be the major organ of glutamine uptake in severe infection; studies in endotoxemic rodents have shown net hepatic glutamine uptake to increase by as much as 8- to 10-fold. This increase is due partially to increases in liver blood flow, but also to a three- to fourfold increase in hepatocyte System N activity in the liver. Cytokines and glucocorticoids mediate the increased uptake of glutamine by the liver in septic states as well as other compounds. Sepsis does not appear to induce an increase in System N gene expression, indicating that the increase in hepatic glutamine transport observed during severe infection is probably regulated at the protein level. The bowel displays a decrease in glutamine utilization during sepsis, a response that may be related to the decrease in circulating insulin-like growth factor-1 (IGF-1) levels that is characteristic of sepsis. Recent studies suggest that IGF-1 has a direct effect on stimulating glutamine transport across the gut lumen and thus may represent a therapeutic avenue for improving gut nutrition during severe infection. The cells of the immune system (lymphocytes, macrophages) are also major glutamine consumers during inflammatory states in which cell proliferation is increased. Under these conditions, glutamine availability can become rate limiting for key cell functions, such as phagocytosis and antibody production.

Pretreatment with SR48692 has different effects on central neurotensin-induced gastric mucosal defense and inhibition of gastric acid secretion in rats.

Neurotensin is a tridecapeptide present in the brain and gastrointestinal tract. Administration of neurotensin into the brain results in responses in the gastrointestinal tract, suggesting a role for neurotensin in the interrelationships that comprise the brain-gut axis. Intracerebroventricular (i.c.v.) administration of neurotensin protects the gastric mucosa against injury caused by cold water restraint (CWR) and also inhibits gastrin-stimulated gastric acid secretion. The hypothesis tested was that these two actions of neurotensin are mediated via its high-affinity receptor. Rats were given neurotensin (60 microgram, i.c.v.) prior to CWR or pylorus ligation after pretreatment with SR48692, a nonpeptide antagonist of the high-affinity neurotensin receptor (0.25 or 2.5 microgram, i.c.v., or 10, 100, or 500 microgram kg-1, i.p.). Neurotensin reduced cold water restraint (CWR)-induced gastric mucosal injury and inhibited gastrin-stimulated acid secretion. Pretreatment with SR48692 (2.5 microgram, i.c.v., or 100 microgram kg-1, i.p.) prior to CWR blocked neurotensin's protection of the gastric mucosa against injury. In contrast, pretreatment with 2.5 microgram SR48692, i.c.v., did not block neurotensin-induced inhibition of acid secretion, whereas 500 microgram kg-1, i.p., partially blocked the inhibition. SR48692 (2.5 microgram, i.c.v.) inhibited acid secretion, suggesting that SR48692 has agonist activity in this system. These results suggest that central neurotensin protects the gastric mucosa against CWR-induced injury via its high-affinity receptor. The receptor that mediates central neurotensin-induced inhibition of gastric acid secretion does not appear to be the high-affinity receptor since the neurotensin receptor antagonist SR48692, when given i.c.v., had agonist activity, inhibiting stimulated acid secretion. High-affinity neurotensin receptors in the periphery appear to play a role in inhibition of stimulated gastric acid secretion.

Regulation of expression of human SP-A1 and SP-A2 genes in fetal lung explant culture.

Human pulmonary surfactant protein A (SP-A) is genetically complex and its regulation may also be complex, reflecting genotypic variability. Fetal lung explants were used to study the regulation of the SP-A genes, SP-A1 and SP-A2, by dexamethasone, interferon, gamma (IFN gamma), cyclic 3',-5' adenosine monophosphate (cAMP), and tumor necrosis factor alpha (TNF alpha). For comparison, the mRNA levels of surfactant protein B (SP-B) and its response to test substances were also examined. Results showed: (a) In control culture total SP-A mRNA varied widely among explants (C.V. = 0.70) compared with SP-B (C.V. = 0.26) (b) IFN gamma significantly increased total SP-A mRNA but there were marked differences among fetal lungs in response to all treatments. (c) SP-A1 mRNA concentration is higher than SP-A2 in both control and treated explants. (d) SP-A1 alleles are inhibited to a greater degree by dexamethasone than SP-A2 alleles. The relative effect of cAMP and IFN gamma on SP-A1 and SP-A2 mRNA varied widely among explants. We conclude that SP-A genotype may account in part for the marked differences in SP-A mRNA concentration among fetal lungs and that the SP-A genes and/or alleles may be differentially regulated.

Mesolimbic expression of neurotensin and neurotensin receptor during stress-induced gastric mucosal injury.

Neurotensin is a neurotransmitter present in the brain and gastrointestinal tract. Intracerebroventricular injection of neurotensin protects rats from gastric mucosal injury caused by cold water restraint (CWR). Direct injection of neurotensin into the nucleus accumbens (NACB), part of the mesolimbic dopamine system, reduces gastric mucosal injury, suggesting that neurotensin confers protection on the mucosa through interaction with the mesolimbic system. The hypothesis is that the concentration of neurotensin in the mesolimbic system decreases during CWR, affecting the expression of neurotensin and the neurotensin receptor. After 1 h of CWR, neurotensin concentration significantly decreased 41% in the NACB and returned toward control concentrations after 2 h of CWR. The concentration of neurotensin mRNA significantly decreased 46% after 1 h CWR and returned toward control after 2 h. In contrast, neurotensin binding sites in the NACB increased from 159 to 228 fmol/mg protein after 1 h of CWR and increased significantly to 280 fmol/mg protein after 2 h CWR, whereas the level of neurotensin receptor mRNA significantly decreased 51 and 50% at 1 and 2 h, respectively. These studies show that neurotensin concentration within the mesolimbic system is transiently reduced by CWR stress and that the number of neurotensin binding sites increases, presumably in response to the decrease in neurotensin.

Effect of genotype on the levels of surfactant protein A mRNA and on the SP-A2 splice variants in adult humans.

Human pulmonary surfactant protein A (SP-A) is encoded by two genes, SP-A1 and SP-A2, that exhibit coding sequence (allelic) and 5' splicing variability. In this report we determine the effect of the genetic variability within the SP-A1 and SP-A2 genes on the level of SP-A mRNAs and on the SP-A2 splicing variants in different individuals. We analysed mRNA specimens from 23 unrelated adults using genotype analysis, Northern analysis and primer extension, and made the following observations. (1) The level of SP-A mRNA varies among individuals (coefficient of variation = 0.49). One SP-A genotype (6A(2)6A(2)1A(0)1A0) appears to be associated with a low to moderate level of SP-A mRNA. (2) The SP-A1/SP-A2 mRNA ratio varies among individuals, from 0.94 (lowest) to 6.80 (highest) within the study population. One genotype appears to be associated with a moderate to high SP-A1/SP-A2 mRNA ratio and another with a low to moderate ratio. (3) There is no correlation between the level of SP-A mRNA and the SP-A1/SP-A2 mRNA ratio. (4) Variability in the ratio of the major SP-A2 splice variants among individuals results from nucleotide differences in the splice-recognition sequence of specific SP-A2 alleles. The SP-A mRNA levels, the SP-A1/SP-A2 mRNA ratio, and the ratio of the major SP-A2 splice variants have a genetic basis in that they vary depending upon the specific SP-A alleles present.

Human SP-A locus: allele frequencies and linkage disequilibrium between the two surfactant protein A genes.

Two surfactant protein A (SP-A) genes and several alleles for each SP-A locus have been previously described. In this report we investigate the potential usefulness of the SP-A loci as markers for genetic studies. We establish conditions that allow the identification of alleles with very similar sequences; We also determine the degree of polymorphism for each SP-A locus: The heterozygosity and polymorphism information content (PIC) values for the SP-A1 locus are 0.63 and 0.55, respectively, and for the SP-A2 locus are 0.50 and 0.56. In the course of these studies, we identify one new SP-A2 allele and show that the SP-A1 and SP-A2 loci are in linkage disequilibrium (P < 0.000001). We also identify 19 of the 20 possible haplotypes in a population of n = 239. Nine of the observed haplotypes reach statistical significance (P < 0.01) in this population, and the segregation of two haplotypes (6A2/1A0 and 6A4/1A) without recombination is verified in a family pedigree. These data together indicate that both SP-A loci are sufficiently polymorphic to be good markers for use in genetic studies. Furthermore, the finding of one novel allele suggests that additional unknown SP-A alleles are yet to be found.

Translation in vivo of 5' untranslated-region splice variants of human surfactant protein-A.

Transcripts of human SP-A genes, SP-A1 and SP-A2, undergo alternative splicing of 5' untranslated-region exons. We reverse-transcribed and amplified free cytoplasmic and polysome-bound RNA and showed that (a) all splice variants of both genes are translated in vivo, (b) the relative translatability of splice variants can differ among individuals, and (c) the relative levels of different SP-A splice variants differ among individuals.

Human SP-A: then and now.

In the short span of ten years, our understanding of human surfactant-associated protein A (SP-A) has advanced rapidly at both the level of the protein and the level of the gene. In the period 1984-1988, the protein was biochemically characterized and two SP-A precursors were identified. The molecular characterization was begun with the publication of an SP-A genomic sequence and sequences of two SP-A cDNAs, suggesting the presence of two SP-A genes. In the period 1991-1992, an SP-A pseudogene, a second SP-A genomic sequence, and an SP-A allelic variant were described. Since that time, a picture of increasing complexity has emerged from studies of the two SP-A genes. This complexity includes alternative splicing of 5' untranslated exons, allelic variants of both SP-A genes, and sequence heterogeneity within the 3' untranslated region. The challenge for the future will be to discover the physiological significance of the genetic complexity of human SP-A.

5' splicing and allelic variants of the human pulmonary surfactant protein A genes.

Human pulmonary surfactant protein A (SP-A) is encoded by two genes, SP-A1 and SP-A2. Reports from our laboratory and other investigations have shown heterogeneity in both genes within three regions (the 5' untranslated [5' UT], the coding, and the 3' untranslated [3' UT] regions). To more fully examine the variability in these regions and characterize the transcription start site in each gene, we used primer extension and 5' RACE to clone and then sequence cDNA clones from two individuals. These cDNAs extended from the transcription start site to approximately 40% of the 3' UT segment. The in vitro translatability of selected cDNAs was also tested. After analysis of our data, we found that: (1) the 5' UT of SP-A genes contains four (A, B, C, D for SP-A1) or three (A, B, D for SP-A2) untranslated exons, three of which (A, B, D) vary in length, and one of which (C) is new; (2) these exons are alternatively spliced and the major splice patterns as well as their relative frequency vary between the two genes (the major pattern for SP-A1 is AD'[81%] and the major patterns for SP-A2 are ABD [44%] and ABD'[49%]); (3) the SP-A1 gene uses three transcription start sites with equal frequency, whereas the SP-A2 gene uses only one; (4) splicing variability occurs among alleles and among individuals; (5) three previously undescribed alleles exist for the SP-A1 gene (6A2, 6A3, 6A4) and two for the SP-A2 gene (1A1, 1A2); and (6) a core group of 10 invariant nucleotides and four invariant amino acids can be used to discriminate between SP-A1 and SP-A2 alleles.

Dinucleotide repeats in the human surfactant protein-B gene and respiratory-distress syndrome.

Pulmonary surfactant, a lipoprotein complex, is essential for normal lung function, and deficiency of surfactant can result in respiratory-distress syndrome (RDS) in the prematurely born infant. Some studies have pointed towards a genetic contribution to the aetiology of RDS. Because the surfactant protein B (SP-B) is important for optimal surfactant function and because it is involved in the pathogenesis of pulmonary disease, we investigated the genetic variability of the SP-B gene in individuals with and without RDS. We identified a 2.5 kb BamHI polymorphism and studied its location, nature and frequency. We localized this polymorphism in the first half of intron 4 and found that it is derived by gain or loss in the number of copies of a motif that consists of two elements, a 20 bp conserved sequence and a variable number of CA dinucleotides. Variability in the number of motifs resulting from either deletion (in 55.3% of the cases with the variation) or insertion (44.7%) of motifs was observed in genomic DNAs from unrelated individuals. Analysis of 219 genomic DNAs from infants with (n = 82) and without (n = 137) RDS showed that this insertion/deletion appears with significantly higher frequency in the RDS population (29.3 as against 16.8%, P < 0.05).

Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver.

Eukaryotic initiation factor (eIF)-2B was purified to greater than 95% homogeneity from both rat and bovine liver. The purified protein consisted of five nonidentical subunits with apparent molecular weights ranging from 30.9 to 89.1 kDa. The holoprotein was characterized in terms of its Stokes radius and frictional coefficient. The isoelectric points for the beta-, gamma-, and epsilon-subunits were found to be 6.4, 6.9, and approximately 6.0, respectively; the alpha- and delta-subunits did not focus well because their isoelectric points as predicted by the nucleotide sequences of cDNAs for the two proteins are greater than 8.5. The purified protein was used as antigen to generate monoclonal antibodies to the epsilon-subunit. The eIF-2B epsilon monoclonal antibodies and monoclonal antibodies to the alpha-subunit of eIF-2 were then used to directly quantitate the amounts of eIF-2B and eIF-2 in rat liver and rat reticulocytes. The ratio of eIF-2B to eIF-2 was found to be approx. 0.6 and 0.3 in liver and reticulocytes, respectively, supporting the proposition that phosphorylation of only part of the total cellular eIF-2 could potentially sequester all of the eIF-2B into an inactive eIF-2.eIF-2B complex. The purified protein was also used as substrate in protein kinase assays. Extracts of rat liver were shown to contain protein kinase activity directed toward the epsilon-subunit, but no other subunit of eIF-2B. Overall, the studies presented here are the first to show a direct quantitation of eIF-2 and eIF-2B in different tissues. They also provide evidence that the epsilon-subunit of eIF-2B is the only subunit of eIF-2B that is phosphorylated by protein kinase(s) present in extracts of rat liver.

Regulation of eukaryotic initiation factor-2 expression during sepsis.

Protein synthesis is stimulated at the level of peptide chain initiation in livers from rats with a sterile or septic abscess. In contrast, peptide chain initiation is inhibited in fast-twitch skeletal muscles from septic rats. We investigated the possible mechanisms responsible for these differential changes in peptide chain initiation between liver and skeletal muscle during sepsis by measuring the cellular content of eukaryotic initiation factor-2 (eIF-2), the extent of phosphorylation of the alpha-subunit of eIF-2, and the activity of eIF-2B. In skeletal muscle, neither the eIF-2 content nor the extent of phosphorylation of eIF-2 alpha was altered during sepsis. However, a significant decrease (P < 0.001) in eIF-2B activity was observed in fast-twitch muscles. In liver, neither the extent of phosphorylation of eIF-2 alpha nor the activity of eIF-2B was different in rats with a sterile or septic abscess compared with control. However, the amount of eIF-2 in liver was increased in both sterile inflammation and sepsis. The relative abundance of eIF-2 alpha mRNA was not increased in either condition compared with control. Analysis of the distribution of eIF-2 alpha mRNA from control rats revealed that only approximately 40% of the message was associated with polysomes. Sterile inflammation or sepsis caused a 50% increase in the proportion of eIF-2 alpha mRNA associated with the polysomes compared with control.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of eukaryotic initiation factors eIF-2, eIF-2B and eIF-2 alpha kinase from bovine liver.

Eukaryotic initiation factors 2 and 2B (eIF-2; eIF-2B) are components of the rate-limiting step in the initiation of eukaryotic protein synthesis and are involved in the regulation of this process. When the alpha-subunit of eIF-2 is phosphorylated by an eIF-2 alpha kinase, the phosphorylated eIF-2 alpha (eIF-2 alpha(P)) binds tightly to eIF-2B and prevents the recycling of eIF-2.GDP to eIF-2.GTP which is required for sustained initiation of protein synthesis. The minute quantities of these proteins which are present in rat liver and muscle cytosol along with hundreds of other proteins has hindered purification efforts, as well as structure:function and regulatory studies. Therefore, procedures were developed for the simultaneous purification of eIF-2, eIF-2B and eIF-2 alpha kinase from kilogram quantities of fresh bovine liver. Briefly, the 0-45% ammonium sulfate precipitate of the 200,000 x g supernatant was solubilized and chromatographed on DEAE-cellulose, heparin-agarose, Mono Q, Mono S, and Superose columns. The availability of purified quantities of these factors will be useful for investigations of molecular mechanisms of action and antibody production.

Conservation analysis of rat and human SP-A gene identifies 5' flanking sequences of rat SP-A that bind rat lung nuclear proteins.

As an initial step toward understanding regulation of tissue-specific expression of SP-A, 5' flanking sequences of the rat SP-A gene and human SP-A I gene were cloned, sequenced, and compared using dot matrix analysis. Two regions were identified, each with a considerable degree of homology between the two species. One region was proximal to the TATAA box, at position -225/-17 in rats and -226/-36 in humans, and the other at position -1115/-1026 in rats and -938/-851 in humans. Studies in rats revealed the specific binding of rat lung nuclear proteins to each of the conserved 5' flanking regions identified in rat SP-A. Binding studies using the rat proximal (rPPS) or distal (rDPS) promoter segments, or overlapping fragments of these segments, with rat nuclear extracts detected the presence of a number (1-4) of lung-specific DNA/protein complexes. When nuclear proteins from liver, a nonexpressing tissue, were used the binding profile of certain nuclear proteins differed from that of the lung. These studies, taken together, suggest that sequences within identified conserved DNA segments in the 5' flanking region of the rat SP-A gene contribute to its tissue-specific expression in rats.

Regulation of eukaryotic initiation factor-2B activity in muscle of diabetic rats.

Peptide-chain initiation is inhibited in fast-twitch skeletal muscle, but not heart, of diabetic rats. We have investigated mechanisms that might maintain eukaryotic initiation factor (eIF)-2B activity, preventing loss of efficiency of protein synthesis in heart of diabetic rats but not in fast-twitch skeletal muscle. There was no change in the amount or phosphorylation state of eIF-2 in skeletal or cardiac muscle during diabetes. In contrast, eIF-2B activity was decreased in fast-twitch but not slow-twitch muscle from diabetic animals. NADP+ inhibited partially purified eIF-2B in vitro, but addition of equimolar NADPH reversed the inhibition. The NADPH-to-NADP+ ratio was unchanged in fast-twitch muscle after induction of diabetes but was increased in heart of diabetic rats, suggesting that NADPH also prevents inhibition of eIF-2B in vivo. The activity of casein kinase II, which can phosphorylate and activate eIF-2B in vitro, was significantly lower in extracts of fast-twitch, but not cardiac muscle, of diabetic rats compared with controls. The results presented here demonstrate that changes in eIF-2 alpha phosphorylation are not responsible for the effect of diabetes on eIF-2B activity in fast-twitch skeletal muscle. Modulation of casein kinase II activity may be a factor in the regulation of protein synthesis in muscle during acute diabetes. The activity of eIF-2B in heart might be maintained by the increased NADPH/NADP+.

The identification and sequence of the actin-binding domain of human red blood cell beta-spectrin.

The junctions of the red blood cell membrane skeleton are formed by interactions between spectrin and actin protofilaments. A spectrin tryptic peptide of 16.5-kDa apparent molecular mass (based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) which binds to F-actin in cosedimentation experiments has been identified. The peptide has been partially purified by gel filtration, anion-, and cation exchange chromatography. Intact spectrin heterodimer causes half-maximal inhibition of the 16.5-kDa peptide/F-actin interaction at a concentration of 5 microM. Comparison of the two-dimensional iodopeptide maps of the 16.5-kDa peptide with maps of alpha- and beta-spectrin, demonstrate that the peptide is generated from the beta subunit. It shows no significant relationship to the peptide maps of the beta-spectrin domains I-IV. Protein sequencing indicated that this actin-binding domain represents a stretch of amino acids at the N terminus of the beta subunit from alanine 47 probably through lysine 186. The sequence derived molecular weight of this actin-binding domain is 16,290 g/mol. The sequence presented represents the region of greatest homology among the spectrin supergene family (spectrin, dystrophin, alpha-actinin).