Effect of repeated stress on a number of plasma amino acids and related compounds in young and old rats

A number of amino compounds were studied in the plasma of young and old rats exposed to an immobilization stress on 3 different days. Baseline values were similar for all compounds between age groups except for slightly higher phenylalanine levels in old animals. Most differences were seen in the stress responses. Stress response intensities for alanine, aspartic acid, methionine, phenylalanine, and tyrosine were generally higher in old rats and recovery times from stress were usually longer. Although the stress responses decreased in young rats for some compounds over the three exposures, old rats showed no or less adaptation during this time. All the other amino acids showed no marked age effects. This study shows that certain plasma amino acids can serve as selective indicators of biological age and that their stress responses might be better markers of the aging process than their resting levels.     

Effects of amino acids on alcohol intake in stroke-prone spontaneously hypertensive rats

The concentration-dependent effects of several PCB, PCDD, and PCDF congeners and several commercial PCB preparations as antiestrogens were determined in the aryl hydrocarbon (Ah)-responsive MCF-7 human breast cancer cell lines. The inhibition of the 17 beta-estradiol-induced secretion of the 52-kDa protein (procathepsin D) was measured using a combination of polyacrylamide gel electrophoresis, double-staining of the protein bands with ISS ProBlue and silver stain, and quantitation by densitometric analysis. For the PCBs, the order of antiestrogenic potency was 3,3',4,4',5-pentachlorobiphenyl > 3,3',4,4',5,5'-hexachlorobiphenyl approximately 3,3',4,4'-tetrachlorobiphenyl > 2,3,3',4,4',5'-hexa, 2,3,3',4,4'- and 2,3,4,4',5-pentachlorobiphenyl > Aroclors 1221, 1232, 1248, 1254, and 1260 were inactive as antiestrogens at the highest concentrations used in this study (10(-6) M). For the PCDDs and PCDFs, the order of antiestrogenic potency was 2,3,7,8-tetrachlorodibenzo-p-dioxin > 2,3,7,8-tetrachlorodibenzofuran > 2,3,4,7,8-pentachlorodibenzofuran > 1,2,3,7,9-pentachlorodibenzofuran > 1,3,6,8-tetrachlorodibenzofuran. With few exceptions, the order of potency for all these congeners and mixtures paralleled their relative activities as agonists for other Ah receptor-mediated responses and their competitive binding affinities for the Ah receptor. The results of this study support the role for the Ah receptor in mediating the inhibition of the 17 beta-estradiol-induced secretion of the 52-kDa protein in MCF-7 cells and also points out the utility of this technique as a bioassay for this class of compounds.     

Effects of various amino acids on gastric lesions induced by acetylsalicylic acid (ASA) and gastric secretion in pylorus-ligated rats

The simultaneous oral administration of various amino acids such as L-lysine, L-arginine, L-histidine, L-serine and others at 750, 250 or 83.3 mg/kg in pylorus-ligated rats produced a marked prevention of the gastric mucosal damages caused by oral acetylsalicylic acid (ASA) at 100 mg/kg. In regard with L-lysine and L-arginine, it was assumed that these amino acids might inhibit the ASA-induced gastric lesions through neutralization of acid because of the high alkalinity of these amino acids. In addition, the lesser effect of the hydrochoride salts of these amino acids as compared with the free form on ASA-induced gastric lesions was observed. The other effective amino acids markedly prevented the back diffusion of acid in response to ASA, suggesting as one of the possible mechanisms of lesion formation. However, L-cysteine, which exerted insignificant effect on ASA-induced gastric lesions, also prevented the back diffusion of acid even though the Na+ concentration had not returned to the control level.     

Release of amino acids and related compounds from the adrenal equivalent and caudal vein of the eel in vitro

The release of catecholamines and cortisol from the perifused adrenal region and caudal vein of the eel (Anguilla rostrata) was compared with the release of 39 amino acids and related compounds. Dopamine, norepinephrine and epinephrine were present in all perifusates of the adrenal region. Dopamine release from the caudal vein exceeded that from the adrenal region, and norepinephrine and epinephrine were not detected. Cortisol was present in the perifusate of the adrenal region but virtually absent in caudal vein perifusate. Of the six substances with known or suspected neurotransmitter function, taurine, aspartate, glutamate, glycine and alanine were present in all or almost all samples from both the adrenal equivalent and the caudal vein. gamma-aminobutyric acid (GABA) was detected in a few samples from either preparation. The release of taurine and phosphoethanolamine may be linked to that of norepinephrine and epinephrine. Adrenocorticotropic hormone (ACTH) enhanced the release of cortisol, aspartate, valine, leucine and ornithine from the adrenal region, but the release appears to be from differing sources or cellular pools. Overall, the study revealed that both the adrenal region and caudal vein release a large number of amino acids and related substances. The caudal vein, and possibly other blood vessels as well, may be a major source of circulating dopamine.     

Effects of L-DOPA/carbidopa administration on the levels of L-DOPA, other amino acids and related compounds in the plasma, brain and heart of the rat

Rats were treated intraperitoneally with a mixture of 250 mg/kg L-DOPA and 40 mg/kg carbidopa or with vehicle and sacrificed 30 min later. Plasma, heart and cortex, midbrain, brainstem and cerebellum were removed from each animal and assayed by HPLC for L-DOPA and a large number of amino acids and related amino compounds. L-DOPA levels increased from undetectable (<0.2 nmol/ml or g) to 1,146, 1,007, 399, 376, 368 and 850 nmol/ml or g in the above tissues. In addition, several amino compounds were significantly affected by L-DOPA/carbidopa (p < or = 0.01). Plasma concentrations of phosphoserine, oxidized glutathione, citrulline, phenylalanine, tyrosine and 1-methylhistidine increased and arginine, glutamic acid and lysine decreased. In the heart, concentrations of phosphoserine, taurine, reduced glutathione, threonine, serine, glutamine, glycine, alanine, valine, GABA, ethanolamine, ammonia and arginine decreased. In the cortex, camosine and homocarnosine increased. In the midbrain, valine increased and leucine, ornithine and oxidized glutathione decreased. In the cerebellum, citrulline increased. In the brainstem, threonine, serine, asparagine, glutamine, oxidized glutathione, alanine, and leucine decreased. In the brainstem, arginine was slightly decreased with a concomitant increase in citrulline (p < 0.05), indicative of nitrous oxide formation. These results show that administration of L-DOPA/ carbidopa not only raises dopamine levels but can also affect other biochemicals and that the observed changes in amino acids and related compounds can perhaps contribute to the beneficial and/or adverse effects of L-DOPA/carbidopa therapy of Parkinson's disease.     

Unidirectional influx of glutamine and other neutral amino acids into liver of fed and fasted rat in vivo

The fractional extraction of unidirectional influx of several neutral amino acids (glutamine, leucine, alanine, tryptophan, and cycloleucine) into rat liver in vivo is studied with a tissue-sampling, single-injection technique. Liver uptake of 14C-amino acid is expressed as an index relative to the hepatic clearance of a 3H-labeled water (3HOH) internal reference. The maximal fractional extraction of 3HOH influx into liver, 0.85, and the rate constant of 3HOH exodus back to blood, 0.87 min-1, provide an estimate of portal blood flow, 0.93 ml min-1 g-1, in the barbiturate-anesthetized, laparotomized rat. Given the extraction data for the 3HOH reference, liver uptake indices for the five amino acids studied are converted into maximal fractional extractions of amino acid influx into liver: glutamine, 0.72 +/- 0.03; leucine, 0.56 +/- 0.02; alanine, 0.43 +/- 0.04; tryptophan, 0.40 +/- 0.03; cycloleucine, 0.25 +/- 0.01; and sucrose, 0.09 +/- 0.02. The influx of glutamine and cycloleucine is shown to be increased (35%) with 48 h of fasting. These data indicate that glutamine penetrates the liver cell membrane faster than any of the 19 amino acids studied thus far. The rate of unidirectional influx of glutamine and other amino acids into liver is estimated and reveals that the capacity of liver cells to transport amino acids is severalfold greater than that of other organs such as brain or muscle.     

Effect of osmotic stress on tissue free amino acids in Pila virens

Tolerated doses of phalloidin, a toxin from the mushroom Amanita phalloides, protect mice against lethal doses of phalloidin. Resistance is conferred by the 1/10 LD95 of phalloidin and sets in at about 8 hours after the pretreatment.     

Mechanism-based inactivation of serine transhydroxymethylases by D-fluoroalanine and related amino acids

Serine transhydroxymethylase, from lamb or rabbit liver, is known to catalyze slow transamination of D-alanine, but not of L-amino acids, in a tetrahydrofolate-independent reaction. Both enzymes will process the D-isomer of beta-fluoroalanine for alpha, beta-elimination of HF to yield an aminoacrylate-pyridoxal-P-enzyme intermediate. This intermediate partitions between harmless hydrolysis to pyruvate, NH4+, and active enzyme-pyridoxal-P (catalytic turnover) and suicidal enzyme alkylation by covalent modification with an average partition ratio of 40-60 turnovers/inactivation event/monomer unit of this tetrameric enzyme. Enzyme inactivation occurs with stoichiometric incorporation of radioactive label from D-[1,2-14C]fluoroalanine. Titration of enzymic cysteinyl --SH groups with 5,5'-dithiobis(2-nitrobenzoate) indicates loss of 1 --SH group on inactivation. Acid hydrolysis of radioactive-inactive enzyme confirms cysteine residue modification. Treatment of inactive enzyme with 6 M urea, then KBH4, followed by acid hydrolysis yields two radioactive compounds, lanthionine and S-carboxyhydroxyethylcysteine, in about equal amounts. The addition of tetrahydrofolate stimulates both pyruvate production and inactivation to equal extents with about a 200-fold rate acceleration at 0.5 mM tetrahydrofolate to turnover numbers of approximately 120 min-1. The Km for D-fluoroalanine is high, 10-60 mM, and this low substrate affinity suggests D-fluoroalanine will not be a useful in vivo agent for selective inactivation of liver cell serine transhydroxymethylases.     

Correlation of blood and brain amino acids in hypoglycemic and normoglycemic rats

Utilization of gluconeogenic amino acids as a source of energy by brain can occur in starved newborn rats. This capacity is lost later in life as evidenced by changing ratios in blood and brain concentrations between fed and fasted animals.     

Solution nonideality related to solute molecular characteristics of amino acids

By measuring the freezing-point depression for dilute, aqueous solutions of all water-soluble amino acids, we test the hypothesis that nonideality in aqueous solutions is due to solute-induced water structuring near hydrophobic surfaces and solute-induced water destructuring in the dipolar electric fields generated by the solute. Nonideality is expressed with a single solute/solvent interaction parameter I, calculated from experimental measure of delta T. A related parameter, I(n), gives a method of directly relating solute characteristics to solute-induced water structuring or destructuring. I(n)-values correlate directly with hydrophobic surface area and inversely with dipolar strength. By comparing the nonideality of amino acids with progressively larger hydrophobic side chains, structuring is shown to increase with hydrophobic surface area at a rate of one perturbed water molecule per 8.8 square angstroms, implying monolayer coverage. Destructuring is attributed to dielectric realignment as described by the Debye-Hückel theory, but with a constant separation of charges in the amino-carboxyl dipole. By using dimers and trimers of glycine and alanine, this destructuring is shown to increase with increasing dipole strength using increased separation of fixed dipolar charges. The capacity to predict nonideal solution behavior on the basis of amino acid characteristics will permit prediction of free energy of transfer to water, which may help predict the energetics of folding and unfolding of proteins based on the characteristics of constituent amino acids.     

Effects of amino acids and gastric inhibitory polypeptide on insulin release in dogs

Insulin release following intravenous administration of an amino acid solution with and without a simultaneous infusion of varying amounts of porcine gastric inhibitory polypeptide (GIP) was studied in dogs. Group I received a 10-amino acid mixture (300 mosmol/kg iv) at 16.6 ml/min for 1 h; group II, amino acid mixture plus 0.5 micrograms.kg-1.h-1 porcine GIP; group III, amino acid mixture plus 1.0 micrograms.kg-1.h-1 of GIP; group IV (a and b) received either 0.5 or 1.0 micrograms.kg-1.h-1 of GIP alone. Compared to group I, groups II and III had a greater insulin response during the first 30 min of the infusion. Group] IV (a and b) showed no insulin release. Glucose concentrations showed no significant change in all groups. From these results, it is concluded that insulin release after intravenous infusion of an amino acid mixture plus GIP is greater than after amino acids or GIP alone. It appears that this effect is more pronounced in the early phase of insulin release.     

Thermal polymerization of amino acids under various atmospheres or at low pressures

The kinds and proportions of amino acids formed in two simulated prebiotic experiments or detected in hydrolyzed extracts of three extraterrestrial samples were found to polymerize thermally under various atmospheres or at low pressures. Yields, tested properties, and amino acid compositions of the polymers were not influenced by the type of enveloping atmosphere, including two simulated prebiotic atmospheres and five pure gases. However, polyamino acids prepared at low pressure (0.02, 10(-4) atm) were obtained in appreciably greater yield than those synthesized at 1 atm; amino acid composition was somewhat influenced by low pressure. The results indicate that polyamino acids could have been formed thermally under a variety of possible prebiotic atmospheres and on planetary bodies of low atmospheric pressure.     

Electrophoretic investigations of blood serum in fasted rats

An affinity column has been synthesized consisting of p-aminophenyl 1-thio-beta-L-fucopyranoside residues attached to Sepharose 4B through succinylated diaminodipropylamine bridges. Surprisingly, it has been found to bind beta-N-acetylglucosaminidase in the serum of Limulus polyphemus (horseshoe crab). The enzyme is eluted with N-acetyl-D-glucosamine at a concentration of 2 mg/ml and with other sugars at higher concentrations. A highly purified enzyme free from other glycosidases is obtained. The enzyme is not eluted by solutions of salt.     

Effects of excitatory amino acids on cerebral oxygen consumption and blood flow in rat

The oncogene Tpr-Met is a constitutively active form of the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met. It comprises the intracellular moiety of Met linked to the dimerization domain of the nuclear envelope protein Tpr, thus functioning as a constitutively activated Met. HGF/SF is responsible for various biological processes including angiogenesis and wound healing, in which secreted serine protease urokinase-type plasminogen activator (uPA) is implicated. The action of HGF/SF on cells is mediated by the autophosphorylation of Met on two carboxyterminal tyrosine residues, Y1349VHVNATVY1356VNV. The two tyrosine residues provide docking sites for various effector molecules, suggesting that multiple signaling pathways are activated to exert biological effects of HGF/SF [Ponzetto et al., Cell (1994) 77: 261]. We found that Tpr-Met efficiently activates the uPA gene via a SOS/Ras/extracellular signal regulated kinase (ERK)-dependent signaling pathway. Mutation of Y1356, which abrogates GRB2 binding, reduced the induction to half of the control level, while mutation of Y1349 showed little effect on uPA induction, suggesting an important but partly replaceable role for GRB2 in Met-dependent uPA gene induction. Mutation of both Y1349VHV and Y1356VNV into optimal PI 3-kinase sites resulted in a residual induction of about one quarter of the control level, suggesting a potential role for PI 3-kinase. Dose-response analysis of the Tpr-Met showed a biphasic curve. These results suggest that the interplay among different signaling molecules on the receptor is important for full induction of the pathway leading to the activation of the uPA gene.     

Effects of aspirin or basic amino acids on collagen cross-links and complications in NIDDM

OBJECTIVE: To determine if long-term therapy with aspirin or basic amino acids for subjects with NIDDM reduces the severity of clinical complications and/or reduces tissue levels of markers of glycooxidative damage. RESEARCH DESIGN AND METHODS: Subjects with NIDDM were administered either aspirin (100 mg/day) or a combination of basic amino acids consisting of L-arginine (2 g/day) plus L-lysine (0.5 g/day) for 1 year. The study was double-blind and placebo-controlled. The presence and severity of retinopathy, nephropathy, and neuropathy were assessed in all subjects at 4-month intervals, as were serum blood glucose, glycohemoglobin levels, and presence of albuminuria. Collagen cross-linking and collagen glycation were measured in skin collagen obtained by biopsy at the beginning and the end of the study. Skin biopsies were also obtained from age-matched control subjects. RESULTS: Skin samples obtained from NIDDM subjects at the beginning of the study had significantly increased levels of glucitolyllysine, pentosidine, and hydroxypyridinium, as compared with age-matched control subjects. Pentosidine levels were significantly correlated with severity of retinopathy and neuropathy, but not nephropathy. Subjects receiving aspirin, but not amino acids or placebo, had significantly decreased levels of skin pentosidine after 1 year of therapy. CONCLUSIONS: It is concluded that 1) low-dose aspirin may reduce glycooxidative damage in people with NIDDM, and 2) treatment may need to continue for more than 1 year before clinical status improves.     

Effects of inhibitory amino acids on the frequency components in sympathetic nerve discharge

The effects of the GABA antagonist picrotoxin and the glycine antagonist strychnine on the frequency components in sympathetic inferior cardiac nerve activity were observed. Picrotoxin (0.03-1.0 mg/kg) increased power in the 10-Hz component of sympathetic activity and produced a dramatic shift in the rhythm to higher frequencies. Only small changes were noted in the 2- to 6-Hz component. Strychnine produced a small generalized increase in power in both frequency bands in sympathetic activity. These data suggest that GABA may play an important role in the generation and maintenance of the 10-Hz rhythm in sympathetic activity while glycine likely inhibits activity at a site of convergence of the two rhythms in sympathetic activity.     

Calcitonin stimulates glycogenolysis in the liver of fasted rats

The effect of calcitonin (CT) on glycogenolysis in the liver was investigated in fasted rats. The fasting produced a marked decrease in the hepatic glycogen content. Thyroparathyroidectomy (TPTX) significantly prevented the decline in hepatic glycogen by fasting as compared with sham operation. This prevention by TPTX was clearly relieved by the subcutaneous administration of CT (80 MRC mU/100 g body weight). The appreciable effect of the hormone was also observed at the dose of 20 and 40 MRC mU CT/100 g body weight. The present results suggest that CT plays a physiological role in the stimulation of hepatic glycogenolysis after fasting in rats.