Transport of a series of D-phenylalanine-glycine hexapeptides across rat alveolar epithelia in vitro.

The effect of lipophilicity on the absorption of peptides from the lungs was investigated. D-phenylalanine (F)-glycine (G) hexapeptides were synthesised to differ, predominantly, only in their lipophilicity. Rat alveolar type II cells were isolated and cultured on plastic, or polycarbonate filters; by day 6 they had de-differentiated to an alveolar type I-like epithelium. The permeability of the monolayers to the hexapeptides was determined. The hexapeptides were metabolically and chemically stable for greater than 24h in the presence of the cells. They did not adhere to the cell culture plastic and were associated only to a low extent with the cell monolayer. The apical to basolateral permeability coefficients for D-F1G5, D-F2G4, and D-F3G3 were 2.19+/-0.53, 1.75+/-0.42 and 2.20+/-0.56 x 10(-7) cm s(-1) respectively. The permeability of the monolayers to D-F1G5 and D-F2G4 was concentration and direction independent, however for D-F3G3 the monolayer was more permeable in the basolateral to apical direction. There was no correlation between the lipophilicity of the hexapeptides and permeability coefficients: other physicochemical parameters did not predict hexapeptide transport. Lipophilicity does not appear to control the transport of hexapeptides across the alveolar epithelium probably as a consequence of the peptides being transported via the paracellular route.

Homocysteine mediated endothelial cell toxicity and its amelioration.

In response to homocysteine induced toxicity in human umbilical vein endothelial cells, minimal changes in the concentration of cellular protein thiols but substantial changes in the concentration of intracellular soluble thiols were observed. The latter correlated closely with changes in cellular glutathione levels. No correlation existed between cellular glutathione levels and cell viability, whereas a close correlation between NAD+ levels and cell viability was demonstrated. Large decreases in cellular NAD+ occurred in response to homocysteine induced toxicity which were accompanied by the production of single stranded DNA. 3-Aminobenzamide, an inhibitor of poly (ADP-ribose) polymerase preserved cell viability and cellular NAD+ levels. Evidence that DNA synthesis was also compromised was revealed by the decreased capacity of homocysteine treated cells to incorporate deoxyuridine. Radical scavengers were also effective in preventing homocysteine induced toxicity. It is likely that the major threat to cells derives from radicals generated intracellularly. Eicosanoid metabolism and the xanthine oxidase system have been identified as two potential sources of radicals.

Lipid peroxidation and homocysteine induced toxicity.

Homocysteine induced toxicity has been examined in cultures of human umbilical vein endothelial cells. The toxic effects of the amino acid alone and the amino acid plus Cu2+ could be prevented by catalase and decreased by desferal, when either was present in the culture medium. When desferal was allowed to accumulate intracellularly, no significant protection from homocysteine induced toxicity was observed. Even though lipid peroxidation accompanied the toxicity induced by homocysteine and homocysteine plus Cu2+, inhibition of lipid peroxidation in either case had no effect on cell viability. The significance of these results is discussed.

Homocysteine uptake by human umbilical vein endothelial cells in culture.

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.

Transport of L-cystine in human umbilical vein endothelial cells in culture.

The uptake of L-cystine into cultured human umbilical vein endothelial cells has been shown to occur by a Na+-independent system which is inhibited by L-glutamate and L-homocysteine, but not by other amino acids. It is likely that the system transporting L-cystine is shared by L-glutamate. Thiol groups associated with membrane bound components appear to be essential for L-cystine uptake but it is not yet evident whether these constitute an integral part of the transporter per se.

Localization of S-adenosylhomocysteine hydrolase and adenosine deaminase immunoreactivities in rat brain.

Antisera against rat-liver S-adenosylhomocysteine hydrolase (SAH-hydrolase) and calf intestinal mucosal adenosine deaminase (ADA) were raised in rabbits and subsequently used to determine the distribution of the corresponding enzymes in rat-brain using the peroxidase-antiperoxidase immunohistochemical procedure. SAH-hydrolase antigenicity was prominent in the neocortex, hippocampal formation, cerebellum and olfactory tubercle. In the cerebellum, only those cells associated with the Purkinje layer possessed pronounced reactivity with anti-SAH hydrolase. The intense staining present could be correlated mainly with nuclei, the cytosol being stained less intensely. Weak ADA antigenicity was found throughout the brain, but strong antigenic reactivity was associated with neurones in the basal hypothalamus, superior colliculus and in nerve fibres in many regions. Many ADA antigenic neurones and fibres were seen in close proximity to blood vessels. The distribution of ADA antigenicity was also studied in cat and rabbit brain. In cat brain only general staining of tissue occurred with anti-ADA and no intensely stained regions comparable to those seen in rat brain were observed. Rabbit brain showed weak specifically stained neurones only in the superior colliculus. Enzyme assays were also performed to confirm immunohistochemical findings. There appears to be little in common between regions which stained intensely with anti-SAH hydrolase and anti-ADA respectively. The possible implications of these findings are discussed.

Adenosine deaminase and histidine decarboxylase coexist in certain neurons of the rat brain.

The enzymes adenosine deaminase (ADA) and histidine decarboxylase (HDC) were immunocytochemically detected in rat brain. The gross distributions of ADA- and HDC-immunoreactive neurons in the basal hypothalamus were very similar. The superficial layers of the superior colliculus showed only ADA-containing neurons. Using adjacent thin-sections of basal hypothalamus, stained alternately for ADA and HDC immunoreactivity, it was possible to show the two labels localized within the same neurons. These observations imply a relationship between two neurochemically distinct putative neurotransmitter/modulator systems, that of histamine and adenosine.

The effects of S-adenosylhomocysteine and S-adenosylmethionine on some purine- and pyrimidine-metabolizing systems.

The effects of S-adenosylhomocysteine and S-adenosylmethionine on some purine- and pyrimidine-metabolizing systems have been examined. Both compounds were capable of acting as relatively good inhibitors of adenosine deaminase, nucleoside phosphorylase, and adenylate deaminase activities but as relatively poor inhibitors of myokinase and nucleoside monophosphate kinase. The inhibitory effects were freely reversible. 5'-Nucleotidase, orotidine 5'- phosphate, and phosphodiesterase were unaffected. Nucleoside phosphorylase was competitively inhibited by both compounds, whereas mixed inhibitory effects occurred with adenosine deaminase.

Studies on the neurochemical properties of cystathionine.

Following the intracerebral administration of [35S]cystathionine, the synaptosome fraction of rat brain was labelled, the greatest uptake of amino acid being associated with hypothalamus. The uptake of [35S]cystathionine by synaptosome preparations isolated from different regions of brain, was typical of that exhibited by amino acids which are not neurotransmitters. Depolarization of the synaptic membrane had no effect on the efflux of [35S]cystathionine from preloaded synaptosomes. The intracerebral administration of cystathionine resulted in an elevation of the levels of brain cyclic AMP, the effect being particularly evident in the cerebellum. Attempts to reproduce this effect in vitro were unsuccessful.

The molecular defect in a case of (cystathionine beta-synthase)-deficient homocystinuria.

1. Cystathionine beta-synthase activity isolated from fibroblast cultures obtained from the skin of a normal and a homocystinuric individual were both cross-reactive with normal human liver cystathionine beta-synthase antibody. 2. Isoelectric focusing revealed a substantial difference in the isoelectric points of the normal and abnormal fibroblast enzymes. 3. Treatment of purified samples of normal and abnormal fibroblast enzymes with sodium dodecylsulphate followed by polyacrylamide gel electrophoresis indicated that both normal and abnormal enzymes were composed of two sub-units of molecular weights 53000 and 70000. 4. A combination of urea and sodium dodecylsulphate treatment revealed that the respective 53000 molecular weight sub-units were different. 5. It has been concluded that the molecular defect in the case of pyridoxine non-responsive homocystinuria examined in the present investigation arises as a result of an alteration in the structural gene which codes for the lower molecular weight sub-unit of cystathionine beta-synthase.

Studies on the use of skin fibroblasts for the measurement of cystathionine synthase activity with respect to homocystinuria.

The levels of cystathionine synthase have been examined in cultured skin fibroblasts obtained from a patient suffering from pyridoxine-non-responsive homocystinuria and compared with the normal and heterozygous states. Levels of synthase activity were found to vary with time in culture and composition of culture media. The physical properties of normal and abnormal synthase activities were markedly different. It has been concluded that caution has to be exercised when using cell culture methods for the purpose of defining normal, heterozygous and homozygous states with reference to homocystinuria.

Substrate specificity of the L-serine O-sulphate degrading activities of Pseudomonas FR.

1. Two enzyme systems obtained from Pseudomonas FR capable of catalysing the alphabeta-elimination of L-serine O-sulphate exhibit a wide range of substrate specificity. Greatest activity was exhibited towards beta-substituted serine and cysteine derivatives. Enzyme A shows a marked preference for the L-isomeric form and enzyme B shows a preference for D-isomers. 2. The alternative activities were shown to be properties of the same enzyme by inhibition properties and heat denaturation experiments. 3. The assay of enzyme A by a number of alternative substrates at various stages during its purification confirmed the multi-substrate specificity of the system. 4. Growth of Pseudomonas FR on S-methyl-L-cysteine as the sole carbon source also resulted in the induction of enzyme B. Growth patterns and levels of induced enzyme were similar to those obtained when L-serine O-sulphate was employed in comparable circumstances.

Effect of induced elevated plasma levels of homocystine and methionine in rats on collagen and elastin structures.

Young growing rats were intraperitoneally injected with mixtures of homocystine and methionine for several weeks. The growth of the animals was inhibited. After 3 weeks 25% of the rats died and isolation of tail tendon collagen and aorta elastin showed that these proteins were deficient in chemical cross-links. Seventy-five % of the rats survived further injections for another 3 weeks and isolated collagen and elastin were found to be normal in cross-linking. The variability in susceptibility of these rats to homocystine-methionine treatment is discussed in relationship to human homocystinuria. It is speculated that the variability is due to variability in in vivo homocysteine levels.