Branchial carbonic anhydrase (CA) of gills of Chasmagnathus granulata (Crustacea Decapoda)

doi:10.1016/S0305-0491(00)00243-1

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology

Volume 127, Issue 1, 1 September 2000, Pages 85-95

https://doi.org/10.1016/S0305-0491(00)00243-1 Get rights and content

Abstract

The occurrence, localization and response to environmental salinity of carbonic anhydrase (CA) activity were studied in all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). CA activity in all gills appeared to be dependent on salinity. The pattern of distribution of CA activity among gills was different upon transition of C. granulata from osmoionoconformity (more uniform distribution) to hyperregulation (highest activity in posterior gills 6–8). Upon abrupt salinity change a differential response of CA activity occurred among gills which could suggest a differential role of CA in ion transport process in different gills of this crab. Furthermore, CA activity in anterior and posterior gills was found in cytosolic and microsomal fractions, although highest activity appeared to be membrane-associated. Both pools of CA were also strongly influenced by salinity and very sensitive to sulfonamide acetazolamide. The results suggest a differential participation of branchial CA in ionoregulatory mechanisms of C. granulata.

Introduction

Carbonic anhydrase (CA) is a ubiquitous enzyme in vertebrates in which it has been intensively studied, particularly in mammals. On the other hand, the information about occurrence, characteristics and physiological role in invertebrates is still fragmentary.

In decapod crustaceans, CA has been involved in all of the major gill functions such as CO₂ excretion, acid-balance and ionoregulation (Henry, 1988a, Henry, 1988b, Botcher and Siebers, 1993, Botcher et al., 1995). The pattern of distribution of CA activity among gills pairs appears to vary depending on habitat and osmoregulatory behavior of the animal (Henry, 1988a, Henry, 1988b, Henry and Cameron, 1982a, Botcher and Siebers, 1993). In euryhaline species such as Callinectes sapidus (Henry and Cameron, 1982a), Carcinus maenas (Botcher et al., 1990a, Botcher et al., 1990b) Eriocheir sinensis (Olsowoski et al., 1995) and in crayfish Pacifistacus leniusculus (Wheatly and Henry, 1987) CA activity was described to be strongly dependent on environmental salinity. Upon acclimation of individuals to reduced salinity CA activity enhanced in posterior gills whereas appeared not to be affected in anterior gills (Botcher and Siebers, 1993). Furthermore, after an abrupt change to dilute media CA activity has been described to increase in posterior gills seven of C. sapidus (Henry and Cameron, 1982b). However, to our knowledge, only in P. lenisculus the response of CA activity to an abrupt salinity change in each individual gill pair has been studied (Henry and Wheatly, 1988).

Several biochemical, physiological and pharmacological studies support the existence of two pools of CA in euryhaline crabs gills — a cytosolic CA which has been suggested to have an important role in posterior gills ion uptake by providing H⁺ and HCO₃⁻ for apical ion transport systems and a membrane-associated CA probably involved in CO₂ excretion (Henry, 1988a, Henry, 1988b, Botcher et al., 1990a, Botcher et al., 1990b, Botcher et al., 1991, Botcher et al., 1995, Botcher and Siebers, 1993, Onken and Riestenpatt, 1998). The existence and predominance of these two forms of CA appear to vary depending on species. In aquatic and semiterrestrial euryhaline crustacean gills the distribution of the cytoplasmic pool of CA appears to be a function of the euryhalinity of the species and the ion transporting capability of the tissue whereas the presence of membrane-associated CA activity would be related to a high metabolic rate (CO₂ production) and an active lifestyle (Henry, 1991).

In C. maenas the predominant membrane-bound CA has been shown to exhibit biochemical and pharmacological features similar with mammalian membrane-bound CA IV (Botcher et al., 1994).

Chasmagnathus granulata (Grapsidae) is a euryhaline semiterrestrial crab which is found from southern Brazil to Patagonia (Argentina) (Boschi, 1964, Botto and Irigoyen, 1969). In Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina) it is one of the dominant crabs in the outer parts where it is exposed to highly and abruptly variable environmental salinity ranging from 4 to 36‰ (Anger et al., 1994, Spivak et al., 1994).

Although C. granulata has been identified as an hyper–hypo osmotic regulator and it is known to support a wide range of environmental salinity (Mañe-Garzón et al., 1974, Gnazzo et al., 1978, Luquet et al., 1992) its branchial ionoregulatory mechanisms at the biochemical level are poorly understood.

The aim of this work was to determine the occurrence, localization and response to environmental salinity of CA activity in all of the gills of C. granulata from Mar Chiquita lagoon.

Section snippets

Chemicals

4-2(hydroxyethyl)-1-piperazinethane sulphonic acid (Hepes) was from Boehringer (Manheim, Germany); sucrose was from Fluka (Germany); ethylenediamine tetraacetic acid (EDTA), Tris–(hydroxymethylamino-methane) (Tris); ethylene glicol N,N′,N′-tetracetic acid (EGTA), acetazolamide and bovine serum albumin were from Sigma (St. Louis, MO, USA). All chemicals were of analytical grade. All solutions were prepared in glass-distilled water.

Animal collection and maintenance

Crabs were caught from a single area of Mar Chiquita lagoon. Only

Localization and distribution of CA activity in gills of C. granulata

CA specific activity was determined in gills 1+2+3 and each of the gills pairs 4–8 of C. granulata acclimated for 3 weeks to either 35 or 10‰ salinity, salinities to which this crab is usually exposed in its natural environment. In 35‰ salinity CA specific activity was homogeneously distributed among individual gills 4–8 and pooled gills 1+2+3. On the other hand, in individuals acclimated to 10‰ salinity highest CA specific activity (about 90–100 μm H⁺ min⁻¹ per mg protein) was found in

Discussion

Results herein described show the occurrence of CA activity sensitive to salinity in each gills of the intertidal crab C. granulata from Mar Chiquita lagoon (Fig. 1, Fig. 2). The more uniform level of CA activity among gills of C. granulata acclimated to 35‰ salinity when hemolymph osmolality and Na⁺, K⁺ and Cl⁻ concentrations are about the same of external medium (Table 1) is in accordance with that described in the euryhaline crab C. sapidus exposed to conditions in which it behaves as

Acknowledgements

We wish to thank Mabel Garcı́a for her technical assistance during the writing of this paper. This work was in part supported by a grant from the University of Mar del Plata, Argentina.

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Na+/K+-ATPase is combined with biological membrane; CA is divided into two groups, one is named cytoplasmic carbonic anhydrase (CAc) and the other one is named glycosyl-phosphatidylinositol-linked carbonic anhydrase (CAg). CAc exists in cytoplasm and CAg is combined with biological membrane (Jillette et al., 2011;López Mañanes et al., 2000). Except for the difference in distribution, both of the molecular structure of the two kinds of CA contain four primary components: the zinc binding site (Christianson and Alexander, 1989), the threonine-199 loop (Merz, 1990), the substrate association pocket (Krebs et al., 1993) and the proton shuttling mechanism (Tu and Silverman, 1989).
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The transfer of euryhaline crustaceans from full-strength seawater to low salinity results in both a rapid up-regulation of carbonic anhydrase (CA; EC 4.2.1.1) mRNA and a slow induction of CA activity. There is a delay of several days between the two processes, which is attributed to the time required to synthesize new enzyme. These delays may also be due to limitations in the cellular uptake of Zn, which is a required post-translational active site modification to CA. To investigate these processes, the euryhaline crabs, Callinectes sapidus and Carcinus maenas, were acclimated to salinities below their isosmotic points (22.5 and 25 ppt, respectively) for 7 days to activate the physiological and molecular mechanisms of osmoregulation. CA mRNA increased 90-fold in C. sapidus and 2-fold in C. maenas within 6 h; whereas it took 48 h for the initial increases in CA activity (120% and 31%), and 4 to 7 days for new acclimated levels (300% and 100%, respectively). Crabs were then transferred to lower salinities (10 and 15 ppt) to induce further CA activity and to determine if previous increases in CA mRNA reduced the time required for subsequent CA induction. Additionally, the expression of the Zn transporter ZIP1 was examined in C. sapidus at 35 and 22.5 ppt. In both species, prior CA mRNA elevation failed to accelerate the rate of CA induction. Levels of CA mRNA did not change in either crab following transfer from intermediate to low salinity. Taken together, these results show that the timecourse of CA induction at low salinity is not limited by the expression of CA mRNA, but by the synthesis of new enzyme from an existing pool of mRNA. No increases in ZIP1 expression occurred at low salinity, therefore these delays may be due to the limits of cellular Zn uptake.
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The occurrence and characteristics of ouabain-insensitive Na⁺ ATPase activity and the response to environmental salinity of the coexistent Na⁺–K⁺ ATPase and ouabain-insensitive Na⁺ ATPase activities were studied in chela muscle of the euryhaline crab Neohelice (Chasmagnathus) granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). Chela muscle exhibited two ouabain-insensitive Na⁺ ATPase activities (a furosemide-insensitive and a furosemide-sensitive activity). I₅₀ for ouabain-insensitive, furosemide-sensitive Na⁺ ATPase activity was about 1.4 mM. Both ouabain-insensitive, furosemide-insensitive and furosemide-sensitive Na⁺ ATPase activities were weakly affected by pH and showed Michaelis–Menten kinetics (K_m = 0.021 and 0.224 mM, respectively). These characteristics appeared to be quite different from those previously described for Na⁺–K⁺ ATPase activity in chela muscle of this crab. Na⁺–K⁺ ATPase and ouabain-insensitive, furosemide-sensitive Na⁺ ATPase activities appeared to be sensitive to environmental salinity. In crabs acclimated to low salinity (10‰), a salinity at which N. granulata exhibits a strong hyperregulatory capacity, Na⁺–K⁺ ATPase activity was higher (117 ± 26 nmol Pi min^− 1 mg prot^− 1) than in 35‰ salinity (23 ± 6 nmol Pi min^− 1 mg prot^− 1) (a salinity at which this crab is osmoionoconforming). On the contrary, ouabain-insensitive, furosemide-sensitive Na⁺ ATPase activity was higher in 35‰ salinity (108 ±15 nmol Pi min^− 1 mg prot^− 1) than in crabs acclimated to 10‰ salinity (36 ± 11 nmol Pi min^− 1 mg prot^− 1). Ouabain-insensitive, furosemide-insensitive Na⁺ ATPase activity was not affected by acclimation of crabs to low salinity. The response to low salinity suggests that Na⁺–K⁺ ATPase could be a component of muscle regulatory mechanisms at the biochemical level secondary to hyperregulation whereas ouabain-insensitive, furosemide-sensitive activity appeared to be predominant upon osmoconforming conditions. The possible differential functional roles of Na⁺–K⁺ ATPase and ouabain-insensitive Na⁺ ATPase activities in muscle are discussed.
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Citation Excerpt :
As for Na+/K+-ATPase, carbonic anhydrase activity of all gills (anterior and posterior gills) is also dependent on the environmental salinity. The pattern of carbonic anhydrase distribution among gills changes from a more uniform distribution when crabs are osmo- and iono-conforming their body fluids to a highest enzyme activity in posterior gills (6–8) when crabs are hyper-regulating these fluids (Mañanes et al., 2000; Genovese et al., 2005a). Therefore, a differential involvement of the carbonic anhydrase present in anterior and posterior gills in the ionoregulatory mechanisms is suggested to occur in N. granulata.
Neohelice granulata (Chasmagnathus granulatus) is an intertidal crab species living in salt marshes from estuaries and lagoons along the Atlantic coast of South America. It is a key species in these environments because it is responsible for energy transfer from producers to consumers. In order to deal with the extremely marked environmental salinity changes occurring in salt marshes, N. granulata shows important and interesting structural, biochemical, and physiological adaptations at the gills level. These adaptations characterize this crab as a euryhaline species, tolerating environmental salinities ranging from very diluted media to concentrated seawater. These characteristics had led to its use as an animal model to study estuarine adaptations in crustaceans. Therefore, the present review focuses on the influence of environmental salinity on N. granulata responses at the ecological, organismic and molecular levels. Aspects covered include salinity tolerance, osmo- and ionoregulatory patterns, morphological and structural adaptations at the gills, and mechanisms of ion transport and their regulation at the gills level during environmental salinity acclimation. Finally, this review compiles information on the effects of some environmental pollutants on iono- and osmoregulatory adaptations showed by N. granulata.

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Branchial carbonic anhydrase (CA) of gills of Chasmagnathus granulata (Crustacea Decapoda)

Abstract

Introduction

Section snippets

Chemicals

Animal collection and maintenance

Localization and distribution of CA activity in gills of C. granulata

Discussion

Acknowledgements

Anal. Biochem.

Comp. Biochem. Physiol.

TIBS

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

Arch. Biochim. Biophys.

Eur. J. Med. Chem.

Hatching rhythms and dispersion of decapod crustacean larvae in a brackish coastal lagoon in Argentina

Helgolander Meeresunters

Sodium–potassium activated adenosine triphosphatase and cation transport

Los crustáceos decápodos brachyura del litoral bonaerense (R. Argentina)

Boln. Inst. Biol. Mar. Mar del Plata.

Biochemistry, localization and physiology of carbonic anhydrase in the gills of euryhaline crabs

J. Exp. Zool.

Carbonic anhydrase in branchial tissues of osmoregulating shore crab (Carcinus maenas)

J. Exp. Zool.

Localization of carbonic anhydrase in the gills of (Carcinus maenas)

Comp. Biochem. Physiol.

Physiological role of branchial carbonic anhydrase in the shore crab (Carcinus maenas)

Mar. Biol.

Membrane-associated carbonic anhydrase from the crab gill: purification, characterization and comparison with mammalian CAs

Arch. Biochem. Biophys.

Carbonic anhydrase, a respiratory enzyme in the gills of the shore crab (Carcinus maenas)

Helgolander Meeresunters