Behavioral study of chemoreception in the sea starMarthasterias glacialis: Structure-activity relationships of lactic acid,amino acids,and acetylcholine

Behavioral responses of Marthasterias glacialis to low molecular compounds were studied under laboratory conditions. Feeding postures, stomach eversions and locomotion of initially inactive animals can be released with very dilute solutions of lactic acid, neutral 2 and 3 carbon amino acids, L isomers of 4 to 6 carbon neutral amino acids, L-arginine, acetylcholine iodide, and several of their analogues. Hunger was induced by temporary withdrawal of food. Responsiveness to feeding stimuli was controlled with L-cysteine and L-leucine. The lowest behavioral thresholds for the most effective feeding stimuli were 3 X 10(-11) mol/l for both enantiomers of lactic acid, 10(-8) mol/l for L-proline and both enantiomers of cysteine and 10(-7) mol/l for acetylcholine iodide and some of the effective neutral amino acids. The behavioral threshold values for chemical stimuli differed by a factor between 30 and 100 in different sea stars. The test concentration was 3 X 10(-7) mol/l, the level at which L-cysteine elicited a complete feeding response from all the animals. Structure-activity comparison of substances less effective than the control stimulus was thus possible. The behavioral threshold of fully effective substances was determined later. The independence of receptor mechanisms for different substances can be inferred as: L-cysteine controlled responsiveness is not always accompanied by responsiveness to neutral amino acids. Autotomized marthasterias arms crawled after stimulation with lactic acid, cysteine, and acetylcholine iodide but did not respond to the feeding stimuli betaine and L-proline. An animal became inactive if electric shocks were paired with L-proline or L-cysteine emanating from an 'electric' food model.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of free amino acids in osmoregulation of cricket blood (Acheta domesticus)

The blood volume increased during normal feeding, and did not decrease during fasting at the end of the stadium. The unexpectedly high blood volume of starved crickets might be an adaptation to increase chances for moulting via stretch receptor stimulation.The amount of blood amino acids was not changed by feeding, but increased with fasting or starvation. Thus amino acid levels in the blood were not directly related to amino acid input from the gut.The blood protein concentration did not change during starvation, but the amino acid concentration was 33% higher in starved crickets that drank water as opposed to those given saline to drink. Thus amino acid levels in the blood were not related directly to blood protein concentration.The blood amino acid concentration was 19–22 mM/l in response to salt intake by feeding crickets or starved crickets drinking saline. The concentration was 32–38 mM/l when the crickets were fasting prior to and after ecdysis or when starved with water to drink during the time when they would normally be feeding. The increase of amino acids during fasting was due to a proportional increase in all amino acids augmented by a 3 × increase in tyrosine. The increase during salt depleting starvation was due to a doubling of the two predominant amino acids proline and glycine. Proline and glycine were not increased in starved crickets drinking saline, thus starvation was not the reason for the increase. This is the first instance where specific amino acids have been implicated in osmoregulation.     

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).     

Changes in Proline Content Accompanying the Uptake of Zinc and Copper by Lemna minor

Lemna minor L. grown in Hoagland solution containing zinc (10ppm) or copper (5 ppm) for 4 d accumulated high levels of thesemetals. Zinc and Cu accumulation in the test plants was accompaniedby a specific pattern of change in proline content. The lattershowed a steep rise during early stages (peak at 12-24 h) followedby a gradual decline until 96 h of treatment. In a dose-responsestudy, lower metal concentrations induced a sharp rise in prolinelevel with a maximum value at 5 ppm, which declined when theconcentration was further enhanced. There was a correspondencebetween the level of proline and total free amino acids in metaltreated plants. The possibility of proline involvement in tolerancemechanisms to heavy metals has been discussed.Copyright 1993,1999 Academic Press Heavy metals, zinc, copper, proline, total free amino acids, Lemna minor     

Participation of kynurenine and its derivatives in disorders of the cardiac rhythm

The influence of tryptophan derivatives: L-kynurenine, 3-hydroxy-DL-kynurenine, kynurenic, xanthurenic, 3-hydroxy-anthranilic and quinolinic acids has been investigated in isolated frog heart. It has been established that L-kynurenine, 3-hydroxy-DL-kynurenine, xanthurenic acid and quinolinic acid at a concentration of 10(-6)-10(-3) mol/l initiate bradycardia. In some cases xanthurenic and quinolinic acids cause a one-minute cardiac arrest in early diastole. 3-hydroxy-anthranilic acid at a concentration of 10(-5) and 5 X 10(-5) mol/l produces premature beats and attacks of tachycardia. In the experiments, using 10(-6)-10(-3) mol/l of kynurenic acid, no impairment of the cardiac rhythm was observed in the isolated frog heart.     

The accumulation of proline in Prasiola crispa during winter in Antarctica

Samples of Prasiola crispa were collected in Antarctica throughout a 13-month period and analysed for free amino acids by HPLC. There was a marked increase in the levels of proline with the onset of winter, concurrent with a decrease in the other predominant amino acids. In January, proline constituted 1.2 ± 0.1 μ mol (g dry weight)⁻¹. whereas by mid-April it was the major component at 28.4 ± 2.9 μ mol (g dry weight)⁻¹. When winter samples were thawed in a growth cabinet, their proline content declined to 4.3 ± 0.5 μ mol (g dry weight)⁻¹ after 7 days. Measurements of photosynthetic quantum yield indicated that winter samples of P. crispa also recovered photosynthetic activity upon thawing. Amino acids and other solutes are involved in the preservation of photosynthetic activities during freezing and it seems probable that proline is involved in cryoprotection in this species. In summer samples, there was no evidence that proline levels in P. crispa increased with the conductivity of the water in which they were found growing.     

The action of proteolytic enzymes on the glycoprotein from pig gastric mucus.

A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.     

Gustatory sensitivity of the herbivore Tilapia zillii to amino acids

The gustatory sensitivity of the herbivorous fish Tilapia zillii was investigated using electrophysiological techniques. Integrated responses from facial taste nerves were recorded during stimulation by l l amino acids. All compounds proved to be effective stimuli. The relative stimulatory efficacy (RSE) determined at 1 × 10 ⁻³ m indicated that stimuli can be organized into three groups on the basis of similarity of efficacy. Effects of pH which altered the RSE of some amino acids are documented. Amino acid RSE differed to some extent from those of previously studied carnivorous fish. There was concordance between the composition of preferred food, effective feeding stimuli and the peripheral receptor responses. These observations support the hypothesis that peripheral chemosensitivity might serve as a mechanism of feeding specialization in some fish.     

Free amino acids of Nicotiana alata anthers during development in vivo

The variation of free amino acids in anthers and pollen of Nicotiana alata Link et Otto has been examined during pollen development in vivo. The purpose was to find clues for optimizing an in vitro medium for cultivation of haploid plantlets from N. alata pollen. The quantitatively most important amino acids were proline, glutamic acid/glutamine, alanine and aspartic acid/asparagine. These exhibited similar patterns of variation during pollen development: first maximum (0.5 to 1.7 μmol per flower) at mitosis, then decrease during early binucleate stage (0.6 to 2.0 m mol per flower). Other amino acids were found in much smaller quantities (max 0.25 μmol per flower).     

Amino acid transport by the filamentous fungus Arthrobotrys conoides

Uptake of l-valine by germinated spores of Arthrobotrys conoides has all the characteristics of a system of transport that requires an expenditure of energy by the cells. It is dependent on temperature and has an energy of activation of 16,000 cal/mole. Uptake is optimal at pH 5 to 6. l-Valine accumulated against a concentration gradient and is not lost from the cells by leakage or exchange. The process requires energy supplied by the metabolic reactions that are inhibited by catalytic amounts of 2,4-dinitrophenol and azide. The kinetics of the system are consistent with a mechanism of transport that depends on a limited number of sites on the cell surface, and the Michaelis constant for the system is 1.5 x 10(-5) to 7.5 x 10(-5)m. Modification of the amino or carboxyl group abolishes l-valine uptake. The process is competitively inhibited by d-valine, glycine, and other neutral amino acids (K(i) = 1.5 x 10(-5) to 4.0 x 10(-5)m), indicating a lack of stereospecificity, and also indicating that aliphatic side chain is not required for binding with the carrier. The transport system has less affinity for acidic amino acids (glutamic and aspartic acids) than neutral amino acids, and a greater affinity for basic amino acids (histidine, lysine, and arginine). The range of affinity is in the order of 100, as measured in terms of K(i) values for various compounds. The data presented provide suggestive evidence that the uptake by A. conoides of all amino acids except proline is mediated by a single carrier system that possesses an overall negative charge.     

A kinetic study on the interaction of deprotonated purine radical cations with amino acids and model peptides

By use of pulse radiolysis techniques, the radical cations of purine nucleotides have been successfully produced by the SO4- ion oxidation. Time-resolved spectroscopic evidence is provided that the one-electron-oxidized radicals of dAMP and dGMP can be efficiently repaired by aromatic amino acids (including tyrosine and tryptophan) via electron transfer reaction. As a model peptide, Arg-Tyr-AcOH was also investigated with regard to its interaction with deprotonated purine radical cations. The rate constants of the electron transfer reactions were determined to be (1 approximately 5) x 10(8) dm(3) mol(-1) s(-1). These results suggest that the aromatic amino acids in DNA-associated proteins may play some role in electron transfer reactions through DNA.     

Positive net movements of amino acids in the hindlimb after overnight food deprivation contribute to sustaining the elevated anabolism of neonatal pigs

During the neonatal period, high protein breakdown rate is a metabolic process inherent to elevated rates of protein accretion in skeletal muscle. To determine the relationship between hindlimb net movements of essential and nonessential amino acids in the regulation of hindlimb protein breakdown during an overnight fasting-feeding cycle, we infused overnight-food-deprived 10- and 28-day-old piglets with [1-(13)C]phenylalanine and [ring-(2)H(4)]tyrosine over 7 h (during 3 h of fasting and then during 4 h of feeding). Extraction rates for aspartate and glutamate after an overnight fast were 15% and 51% in the 10-day-old compared with 6% and 25% in the 28-day-old (P < 0.05) piglets, suggesting an altered requirement for precursors of amino acids to shuttle nitrogen to the liver as early life progresses. This occurred simultaneously with marginal positive hindlimb net balance of essential amino acids after an overnight fast, with negative net release of many nonessential amino acids, such as alanine, asparagine, glutamine, glycine, and proline. This suggests that newborn muscle does not undergo significant protein mobilization after a short period of fasting in support of an elevated rate of protein accretion. Furthermore, tyrosine efflux from hindlimb breakdown between overnight fasting and feeding periods was not different in the 10-day-old piglets, for which tyrosine was limiting, but when tyrosine supply balanced requirements in the 28-day-old piglet, hindlimb efflux was increased (P = 0.01). The results of the present study indicate that proteolysis and net movements of amino acids are coordinated mechanisms that sustain the elevated rate of net protein accretion during overnight feeding-fasting cycles in the neonate.     

Intestinal apical amino acid absorption during development of the pig

Amino acids originating from the diet are the principal metabolic fuels for the small intestine, and although the developing intestine is exposed to dramatic changes in the types and amounts of protein, there is little known about rates of amino acid absorption across the apical membrane during development. Therefore, rates of absorption were measured for five amino acids that are substrates for the acidic (aspartate), basic (lysine), neutral (leucine and methionine), and imino (proline) amino acid carriers using intact tissues from the proximal, mid-, and distal small intestines of pigs ranging in age from 90% of gestation to 42 days after birth (12 days after weaning). Rates of absorption (sum of carrier-mediated and apparent diffusion) were highest at birth (except for proline) and declined by an average of 30% during the first 24 h of suckling. There were continuing declines for leucine, methionine, and proline but not for aspartate and lysine. Due to rapid growth of the intestine, absorption capacities for all amino acids increased faster than predicted from gains in metabolic mass. Regional differences for rates of absorption were not detected until after birth, and only for aspartate and proline. Maximum rates of saturable absorption (nmol. min(-1). mg tissue(-1)) by the midintestine increased during the last 10% of gestation, were highest at birth, and then declined. The contribution of apparent diffusion to amino acid absorption was lowest at birth, then increased after onset of suckling.     

Intermolecular interaction of adenosine-5'-phosphate with phenylalanine and tryptophan by nuclear magnetic resonance]

Equilibrium constants (K) of the complex formation of adenosine-5'-phosphate with phenylalanine (27 l/mol) and tryptophan (67 l/mol) in water (D2O, pD 9.9) have been determined. The comparison of the equilibrium constant values allowed to find the sequence of probabilities of the approach of aromatic amino acids to adenosine-5'-phosphate (tryptophan greater than tyrosine greater than phenylalanine). It has been shown that the dependence of lg K on l/T in the system phenylalanine-adenosine-5'-phosphate is non-linear. The ranges of the positive values of enthalpy (deltaH) and entropy (deltaS) changes of the complex phenylalanine-adenosine-5'-phosphate formation in the temperature interval 28-65 degrees have been found (deltaH = 0.2-1.5 kcal/mol, delta S = 14-50 e.u.). The conclusion about the hydrophobic interaction of aromatic cycles of nucleotide and amino acids has been deduced from the received data.     

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5mM proline, 10mM glutamine, and 10 or 20mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.     

Free amino acids in claw muscle and haemolymph from Australian freshwater crayfish at different stages of the moult cycle

Amino acids were measured in claw muscle and haemolymph in the freshwater decapod crustacean, Cherax destructor, at different stages of the moult cycle. The total pool of amino acids in muscles from animals in intermoult (97+/-13 mmol kg(-1) muscle), premoult (80+/-20 mmol kg(-1)) and postmoult (97+/-19 mmol kg(-1)) were not significantly different. Despite the relatively stable total pool of amino acids, there were changes in the concentrations of alanine, glutamine and proline over the moult cycle. Compared to intermoult, claw muscles from animals in premoult had a lower concentration of proline, and animals in postmoult had higher concentrations of alanine and glutamine, but lower concentrations of proline. Concentrations of alanine and glutamine in claw muscle of animals in postmoult were higher and proline concentrations lower than in the same animals during the premoult stage. The concentration of proline in haemolymph was lower in animals in premoult and postmoult compared to intermoult. The total amino acid pool in the claw muscle of Cherax destructor did not change significantly over the moult which is distinctly different to the changes in amino acids reported in the claw muscles of marine decapod crustaceans.     

Adenosine triphosphate increases the reactivity of the afferent arteriole to low concentrations of norepinephrine

Adenosine triphosphate (ATP) and norepinephrine (NE) interact in the control of blood flow in the kidney. A combined effect of NE and ATP has not been previously investigated at the level of the afferent arteriole (Af). We studied the effects of ATP on the contractile response of the Af to NE. Vascular reactivity to ATP, NE, and their combination was investigated in isolated perfused Af from mice. The roles of alpha-adrenoceptors and P2-ATP-receptors were investigated by use of specific agonists and antagonists. Cytosolic calcium was measured using the fluorescent calcium dye fura-2. ATP in concentrations from 10(-12) to 10(-4) mol/l induced transient contractions. NE constricted the Af in a dose-dependent manner and induced significant contractions at > 10(-7) mol/l. Treatment with ATP (10(-8) and 10(-6) mol/l) increased the NE response. Diameters were reduced by 20% already at 10(-11) mol/l NE during ATP treatment of 10(-6) mol/l. ATP increased the calcium response to NE significantly at 10(-8) and 10(-7)mol/l NE. The P2-type ATP receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10(-5) mol/l) abolished the sensitization of the NE response by ATP. The alpha(1)-blocker prazosin (10(-7) mol/l) inhibited the ATP effect, as did the alpha 2-blocker yohimbine (10(-7) mol/l). Neither the phenylephrine- nor clonidine-induced concentration response curves was affected by ATP in the bath solution. Costimulation with ATP enhances the response of the Af to NE. This effect is mediated by increased cytosolic calcium. The enhancing effect involves P2-type ATP receptors and both alpha (1)- and alpha 2-adrenoceptors.     

The role of hemolymph proline as a nitrogen sink during blood meal digestion by the mosquito Aedes aegypti

Mosquitoes utilize the amino acids derived from blood meal protein to produce egg proteins. But the amino acids can also be used to produce egg lipid or can be oxidized for energy production. These latter two processes result in the release of nitrogen as toxic ammonia. Therefore, amino acids must be processed in such a way that amino acid nitrogen can be incorporated into non-toxic waste products. Proline is the predominant amino acid in the hemolymph of the adult female mosquito Aedes aegypti. After feeding on albumin meal, hemolymph proline levels increased five-fold over unfed levels, reached maximal levels in the first hours after feeding and remained high through oviposition. Hemolymph proline levels increased as the concentration of protein in the meal increased. When starved of sugar for 24 h prior to feeding on an albumin meal, hemolymph proline levels increased four-fold over the proline levels of non-starved mosquitoes. Proline levels after feeding on a protein deficient in essential amino acids, pike parvalbumin, increased to twice the levels of albumin fed mosquitoes. Based on these observations, we propose that mosquitoes utilize proline as a temporary nitrogen sink to store ammonia arising from deamination of blood meal amino acid.     

Olfactory sensitivity of the herbivorous grass carp, Ctenopharyngodon idella, to amino acids

The olfactory sensitivity of a herbivore, the grass carp, Ctenopharyngodon idella , to ten amino acids was investigated using electrophysiological recording techniques. Underwater electroolfactogram recordings increased exponentially with logarithmic increases of stimulus concentration from threshold to 1 × 10⁻³ m. Calculated detection thresholds ranged from 1 × 10^−7.15 m to 1 × 10^−8.7 M and are similar to those previously reported for carnivorous fish species. The relative stimulatory efficacy (RSE) of the ten amino acids determined at 1 × 10⁻⁴ M revealed five groups of stimuli, with cysteine the most stimulatory and proline the least. Measures of amino acid RSE did not differ significantly from those of previously studied carnivores, except for arginine. Grass carp showed strong responses to this compound, whereas it has produced only limited responses in carnivores. Differences in the responsiveness of herbivores and carnivores were not observed, suggesting that feeding niche segregation is probably not facilitated by differential olfactory sensitivities to feeding stimuli.     

Sources of Proline-nitrogen in Water-stressed Soybean (Glycine max L.) I. Protein Metabolism and Proline Accumulation

Water stress was imposed upon soybean plants (Glycine max L.)grown in a greenhouse by withholding irrigation for 10 daysafter 5 weeks of growth, and the changes under stress in thelevels of free amino acids, free ammonia and protein were determinedin detail. With a decrease in the leaf water potential, theprotein content gradually decreased, whereas the free ammoniacontent was relatively constant. Water stress induced an increasein the levels of free amino acids normally present in proteinsuch as isoleucine, leucine, valine, phenylalanine, glutamineand histidine, indicating that protein hydrolysis occurs understress. Proline accumulated only under severe stress (below–1.5 MPa) and attained 0.86% of the dry weight on day10 (–2.6 MPa). Asparagine also accumulated only undersevere stress (below –2.0 MPa). The concentration of glutamicacid, alanine, aspartic acid, serine, glycine and arginine remainedvirtually unchanged during the stress period. Total proline(protein-bound+free) first decreased during mild to moderatestress, and then increased over that of the well-irrigated controlplants at severe stress due to a remarkable accumulation offree proline. These findings indicate that some de novo synthesisof proline occurs under severe stress and that the nitrogensource for this proline synthesis may be protein. (Received July 4, 1981; Accepted September 11, 1981)