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Strikingly, in spite of its physiological importance, information about occurrence, biochemical characteristics and mechanisms of regulation of aminopeptidase-N (APN) in the hepatopancreas of intertidal euryhaline crabs is still lacking. In this work, we determined the occurrence, biochemical characteristics, response to environmental salinity and dopamine of APN in the hepatopancreas of the euryhaline crab Neohelice granulata (Dana 1851) from the open mudflat of Mar Chiquita coastal lagoon (Buenos Aires province, Argentina). APN activity was maximal at pH and temperature range of 7.6-9.0 and 37-45 °C, respectively. APN activity exhibited Michaelis-Menten kinetics (apparent Km = 0.19 ± 0.10 mM) (pH 7.6, 37 °C) and appeared to be sensitive to bestatin (I 50 = 15 mM) and EDTA (I 50 = 9 mM). In crabs acclimated to 10 psu (hyper-regulation conditions) and 37 psu (hypo-regulation conditions), APN activity was about 45 and 160% higher, respectively, than in 35 psu (osmoconformation). APN activity in the hepatopancreas was stimulated in vitro (about 137%) by 10(-4) M dopamine. Higher dopamine concentrations produced a similar extent of increase. The responses of APN activity to salinity and dopamine in vitro suggest the role of APN in digestive adjustments upon hyper and hypo-regulatory conditions and its modulation via direct mechanisms on hepatopancreas by dopamine.

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Studies on digestive adjustments at the biochemical level in relation to salinity in euryhaline crabs are lacking. Moreover, knowledge of biochemical digestive characteristics of euryhaline crabs (i.e. occurrence and characteristics of key digestive enzyme activities) is still scarce and fragmentary. We studied the occurrence, characteristics and response to low salinity of amylase, maltase and sucrase activities in the hepatopancreas of the euryhaline crab Neohelice (Chasmagnathus) granulata. Maximal amylase and maltase activities were found at pH 5.2. Sucrase activity was maximal within the pH range 3.6-5.2. Amylase, maltase and sucrase activities showed a Michaelis-Menten kinetics (km = 0.41±0.10 mg ml-1 ; 1.37±1.03 mM and 0.55±0.45 mM, respectively). In crabs acclimated to low salinity (10 psu; hyperregulating conditions), amylase activity (7263±980 µg maltose min-1 mg prot-1) was higher than in 35 psu (osmoconforming conditions) (3605±340 µg maltose min-1 mg prot-1). Maltase and sucrase activities (497±98 and 64±16 µg glucose min-1 mg prot-1 , respectively) were similar in both salinities. The response of amylase activity to low salinity suggests a role in digestive adjustments upon hyperregulation. This study contributes to a better understanding of the complexity of the biochemical adaptations to low salinity in euryhaline crabs.

SUMMARY:We studied the occurrence and characteristics of lipase activity and the response of lipase and proteolytic activity to salinity and dopamine injections in hepatopancreas of the euryhaline crab (Cyrtograpsus angulatus). Lipase activity was maximal at pH 8.5; it exhibited Michaelis-Menten kinetics (apparent K m =0.019 mM), was higher at 37°C and appeared to be cold tolerant, being also high at 4°C. In 10 psu (hyper-regulation conditions), lipase and proteolytic activity were about 3 and 5 times higher, respectively, than in 35 psu (osmoconformation). In 40 psu (hypo-regulation), lipase activity was about three times higher than in 35 psu, while proteolytic activity was similar. Lipase activity was inhibited in vivo by 10 -4 M dopamine in 35 psu but not in 10 or 40 psu. Proteolytic activity was not affected by 10 -4 M dopamine. The differential responses of lipase and proteolytic activity to salinity and dopamine suggest the occurrence of distinct digestive adjustments and mechanisms of regulation upon osmoregulatory conditions. This study contributes to a better understanding of the complexity of the biochemical adaptations to salinity in euryhaline crabs. The fact that higher digestive enzyme activities could be associated with a differential digestive capacity potentially leading to enhanced availability of energy substrates is discussed.Keywords: Cyrtograpsus angulatus, euryhaline crabs, salinity, hepatopancreas, digestive enzymes, dopamine, Mar Chiquita lagoon. RESUMEN: EfEcto dE la salinidad ambiEntal y dE la inyEcción dE dopamina sobrE Enzimas digEstivas clavE En hEpatopáncrEas dEl cangrEjo EurihalinoCyrtograpsus angulatus (dEcapoda: brachyura: varunidaE). -Se estudió la existencia y características bioquímicas de actividad de lipasa y la respuesta a salinidad y a dopamina de actividad de lipasa y proteolítica total en el hepatopáncreas del cangrejo eurihalino Cyrtograpsus angulatus. La actividad de lipasa fue máxima a pH 8,5; mostró una cinética michaeliana (K m =0.019 mM); fue mayor a 37°C, y resistente a baja temperatura (4°C). En 10 psu (condición de hiperregulación), las actividades de lipasa y proteolítica fueron 3 y 5 veces mayores, respectivamente, que en 35 psu (condición de osmoconformación). En 40 psu (condición de hiporregulación), la actividad de lipasa fue de alrededor 3 veces mayor que en individuos aclimatados a 35 psu, mientras que la actividad proteolítica fue similar. La actividad de lipasa fue inhibida in vivo por dopamina 10 -4 M en 35 psu pero no fue afectada en 10 o 40 psu. Dopamina 10 -4 M no afectó la actividad proteolítica. Las respuestas de las actividades de lipasa y proteolítica a salinidad y a dopamina permiten sugerir la existencia de ajustes digestivos y mecanismos de regulación diferenciales en relación al estado osmorregulatorio. Los resultados constituyen un aporte relevante al conocimiento sobre las complejas adaptaciones a nivel bioquímico a la salinidad en cangrejos eurihalinos.

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We studied the biochemical characteristics and modulation by dopamine, glucagon and cAMP of lipase activity in hepatopancreas of the euryhaline crab Neohelice granulata (Dana, 1851), considered to be an emergent model for biochemical, physiological and ecological research. Lipase activity was maximum at pH 8.5; it exhibited Michaelis-Menten kinetics (apparent K m = 0.018 mM), was highest at 37°C but appeared to be cold-and heat-tolerant, since it was high at 4°C and at 45°C. Lipase activity was enhanced upon incubation of hepatopancreas with 10~4 M dopamine (about 100%), 2 x 10~3 M glucagon (about 250%) and 10-4 M cAMP (about 150%) suggesting a role of these chemical messengers in mechanisms of regulation of lipolytic activities and its direct effect on the hepatopancreas. The concomitant decrease in triglycerides content upon dopamine and cAMP treatment suggests a link between enhanced lipase activity by these messengers and triglycerides catabolism. RESUMENEl objetivo de este trabajo fue estudiar caracterfsticas bioqui'micas y la modulation por dopamina, glucagon y AMPc in vitro de la actividad de lipasa en hepatopancreas del cangrejo eurihalino Neohelice granulata (Dana, 1851) el cual es considerado un modelo emergente para la realization de estudios sobre bioqulmica, fisiologfa y ecologi'a. La actividad de lipasa fue maxima a pH 8.5, exhibio una cinética de Michaelis-Menten (7Cm = 0.018 mM), el valor mas alto de actividad fue registrado a 37°C y es aparentemente tolerante al frio y al calor ya que mantuvo alta actividad a 4°C y 45°C. La actividad de lipasa en hepatopancreas se incrementô en presencia de dopamina 10" 4 M (100%), glucagon 2 x 10~3 M (250%) y AMPc 10-4 M (150%). La concomitante disminucion 3en el contenido de triglicéridos del hepatopâncreas en presencia de dopamina y AMPc sugiere la existencia de una relacion entre el incremento de la actividad de lipasa y la posible movilizacion de réservas sugiriendo el roi de estas mensajeros qui'micos como componente de las vfas de serialization involucrados en la regulation del metabolismo de lipidos.