Osmotic effect of choline and glycine betaine on the gills and hepatopancreas of the Chasmagnathus granulata crab submitted to hyperosmotic stress

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Abstract

Choline is the precursor of glycine betaine, a compatible osmolyte that can maintain the osmotic balance of cells in high osmolality media. This study examined the effect of hyperosmotic stress on the 14C-choline uptake in the hepatopancreas and gills of Chasmagnathus granulata crabs. Uptake in the hepatopancreas was influenced by different sodium concentrations in the incubation media. A reduction of uptake was observed in the hepatopancreas, anterior and posterior gills, in the presence of increasing concentrations of non-radioactive choline. There was a reduction of choline uptake in the anterior and posterior gills of animals submitted to long-term (72 h) hyperosmotic stress compared to the control group. The hepatopancreas incubated with 14C-choline during long-term hyperosmotic stress presented choline uptake values approximately two times higher than in the control group. The glycine betaine synthesis of this group was higher than the control group. These results demonstrate the osmotic effect of glycine betaine in crabs during hyperosmotic stress, and this effect was only observed in the hepatopancreas, and during the long-term 72-h stress. This paper shows, for the first time, the effect of choline and glycine betaine in the osmoregulatory process in crustaceans.

Introduction

Environmental factors such as drought, salinity and temperature extremes have been limiting factors for species survival. Organisms that live in habitats where these factors are a major issue have developed a few adaptations to survive in these environments. They accumulate organic solutes such as polyhydric alcohols, free amino acids and quaternary ammonium and or tertiary sulphonium compounds in response to osmotic stress. The accumulation of these solutes in response to osmotic stress is a metabolic adaptation found in stress-tolerant invertebrates and vertebrates, suggesting convergent evolution for this trait (Rathinasabapathi, 2000).

Betaine is used as a non-disturbing osmolyte by plants, bacteria, invertebrates and vertebrates to compensate hypertonic stress (Petty and Lucero, 1999). The choline is taken up by a sodium-dependent transport protein. Okuda et al. (2000) demonstrate that the amino acid sequence of the choline transporter in cholinergic neurons of rats has a significant homology with the members of the sodium dependent glucose transporter family (26%). The accumulation of osmotically active compatible solutes, such as glycine betaine, is used by some organisms to ease growth under stress conditions, and also to stabilize macromolecules against salt (hyperosmotic stress), increase urea and denaturation by heat and freezing. In mammals, it is accumulated in the cells of the kidney medulla, where it regulates hyperosmotic stress and prevents denaturation by the urea (Randal et al., 1996, Moeckel and Lien, 1997).

Bacteria show tolerance against hyperosmotic stress by uptake and/or synthesis of the glycine betaine (Van Der Heide and Poolman, 2000). In Bacillus subtilis two genes were identified, whose products are used to convert choline into glycine betaine (Boch et al., 1996). The internal concentration of betaine in A. halophila may represent up to 33% of its dry weight and E. halochloris can show an accumulation of up to 2.5 M of intracellular betaine concentration (Nyyssola et al., 2000).

In invertebrates, the use of this osmolyte is very important, mainly during osmotic regulation processes. This is demonstrated in the marine clam Mytilus californianus, where the betaine transporter present in the gills is sodium-dependent and the activity of this transporter decreases with the reduction of the osmolality (Wright et al., 1992). In the squid Lolliguncula brevis it was found that the activation of betaine transport in hypertonic conditions could affect the regulation of volume and the excitability of motor neurons (Petty and Lucero, 1999). The oyster Crassostrea virginica presents a betaine synthesis from exogenous choline. It was found that mitochondria from oysters adapted to high salinity do take up choline and synthesize betaine faster than at low salinities (Pierce et al., 1995). The glycine betaine synthesis rate is ruled by the rate of choline uptake by mitochondria from Limulus polyphemus. Choline uptake by the mitochondria increases when the ion concentration is higher, and stimulates the metabolic pathway, resulting in an increased glycine betaine production (Dragolovich and Pierce, 1992, Dragolovich and Pierce, 1994). During the adaptation period of hyperosmotic stress, the heart cells of horseshoe crabs accumulated glycine betaine as an active osmolyte. (Dragolovich and Pierce, 1992)

Although free amino acids are an extremely important osmolyte in marine organisms, methylamines in general, and particularly betaine are frequently found at higher amounts in several euryhaline animals (Pierce et al., 1995). In crustaceans, most studies have demonstrated the importance of free amino acids in the osmoregulation process (Gilles, 1982, Gilles, 1983, Gilles, 1997, Gilles and Pequeux, 1985).

The estuarine crab, Chasmagnathus granulata, inhabits salt marshes along the coast of Southern Brazil, Uruguay and Argentina (Boschi, 1964). This crab is a good hyper- and hypo-osmoregulator (Bromberg, 1992, Miranda, 1994, Bromberg et al., 1995, Castilho et al., 2001), and tolerates long-term exposure to freshwater and hypersaline medium (40‰) (Nery and Santos, 1993).

A study performed by Deaton (2001) demonstrated that the concentration of glycine betaine in gills of the Geukensia demissa clam increases significantly in a period of hyperosmotic stress. Morris and Edwards (1995) suggest the possibility that gills are not the only major site of ion absorption during osmotic stress. It would appear that the Na+/K+-ATPase activity of the hepatopancreas is much more responsive to external stimulation (osmotic stress) than the gills, and it becomes the main site of absorption of these ions for osmotic regulation.

Several studies in crustaceans show the importance of hepatopancreas in the management of substrates (Kucharski and Da Silva, 1991, Schein, 1999), nitrogen production (Koening, 1981, Tan and Choong, 1981, Schein, 1999), metabolism (Vinagre and Da Silva, 2002) and PEPCK activity during osmotic shock (Oliveira and Da Silva, 1997, Oliveira and Da Silva, 2000, Schein et al., 2004).

The crustacean hepatopancreas express a variety of transporters (Ahearn et al., 1992), but little attention has been paid to their possible involvement in osmoregulation by euryhaline species.

Although there are many studies concerning choline/glycine betaine, there is no evidence that glycine betaine accumulates in crustaceans as an osmotic solute. And that is the aim of the present study, to determine the effect of hyperosmotic stress on choline uptake and on the glycine betaine synthesis, in hepatopancreas, anterior and posterior gills of the C. granulata crab. Hypothetically, this betaine will then be released by these tissues into the hemolymph and taken up by other tissues to increase the cytoplasmatic osmotic concentration and deal with the higher osmotic concentration of the media.

Section snippets

Animals

Male C. granulata in stage C of the intermolt cycle (Drach and Tchernigovtzeff, 1967) were collected from a lagoon (Lagoa Tramandaí) located in the state of Rio Grande do Sul, Brazil. This is an estuarine environment where these animals are naturally submitted to a salinity range of lower than 1‰ up to 35‰. The animals were collected according to Brazilian environmental laws (Portaria no.: 332/90 IBAMA). Animals weighing 15–17 g were placed in aquaria at a salinity of 20‰, at a temperature of

Uptake of [methyl-14C] choline chloride by the anterior and posterior gills and hepatopancreas of C. granulata

Fig. 1 shows the influence of sodium on the choline uptake in the hepatopancreas. There is a significant rise in the choline uptake with the higher concentration of sodium (400 mM), when compared to the concentrations of 0 and 300 mM. The same experiment was performed in the gills, but no influence of sodium on the choline uptake in this tissue was observed.

The effect of different concentrations of non-labeled choline on the choline uptake in AG and PG is shown in Fig. 2, and that in the

Discussion

The investigation of osmotic stress has been the aim of studies in several organisms such as bacteria, plants, invertebrates and mammals. In the research performed on invertebrates studies were found mainly on mollusks (Pierce et al., 1995, Pierce et al., 1997, Perrino and Pierce, 2000a, Perrino and Pierce, 2000b, Deaton, 2001) and crustaceans (Gilles, 1983, Da Silva and Kucharski, 1992, Morris and Edwards, 1995, Gilles, 1997, Castilho et al., 2001, Schein et al., 2005). The ability to

Acknowledgments

This work was supported by grants from Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. We would like thanks to Gabriel Parmegiani Jahn for the support in the English version of the manuscript. [SS]

References (47)

  • A. Nyyssola et al.

    Extreme halophiles synthesize betaine from glycine by methylation

    J. Biol. Chem.

    (2000)
  • T. Okuda et al.

    Functional characterization of the human high-affinity choline transporter

    FEBS Lett.

    (2000)
  • G.T. Oliveira et al.

    Gluconeogenesis of hepatopancreas of Chasmagnathus granulata crabs maintained on high-protein or carbohydrate-rich diets

    Comp. Biochem. Physiol. A

    (1997)
  • G.T. Oliveira et al.

    Hepatopancreas gluconeogenesis during hyposmotic stress in crabs Chasmagnathus granulata maintained on high-protein or carbohydrate-rich diets

    Comp. Biochem. Physiol. B

    (2000)
  • B. Rathinasabapathi

    Metabolic engineering for stress tolerance: installing osmoprotectant synthesis pathways

    Ann. Bot.

    (2000)
  • V. Schein et al.

    Effect of hyperosmotic shock on phosphoenolpyruvate carboxykinase gene expression and gluconeogenic activity in the crab muscle

    FEBS Lett.

    (2004)
  • V. Schein et al.

    Effects of hypo- or hyperosmotic stress on gluconeogenesis, phosphoenolpyruvate carboxykinase activity, and gene expression in jaw muscle of the crab Chasmagnathus granulata: seasonal differences

    J. Exp. Mar. Biol. Ecol.

    (2005)
  • C.H. Tan et al.

    Effect of hyperosmotic stress on hemolymph protein, muscle nihydrin-positive substances and free amino acids in Macrobrachium rosenbergii (De Man)

    Comp. Biochem. Physiol. A

    (1981)
  • A.S. Vinagre et al.

    Effects of starvation on the carbohydrate and lipid metabolism in crabs previously maintained on a high protein or carbohydrate-rich diet

    Comp. Biochem. Physiol. A

    (1992)
  • G.A. Ahearn et al.

    Invertebrate gut diverticula are nutrient absorptive organs

    Am. J. Physiol.

    (1992)
  • J. Boch et al.

    Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

    J. Bacteriol.

    (1996)
  • E.E. Boschi

    Los crustáceos decapoda brachiura del litoral bonaerense

    Bol. Inst. Biol. Mar del Plata

    (1964)
  • Bromberg, E., 1992. Dinâmica osmo e ionoregulatória de Chasmagnathus granulata Dana, 1851 (Crustacea, Decapoda,...
  • Cited by (0)

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