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.
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.
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.
Left ventricular (LV) hypertrophy (LVH) is an adaptive response to hemodynamic overload, but also contributes to the pathogenesis of heart failure. LVH can be concentric (cLVH) but subsequent dilatation and progression to eccentric hypertrophy (eLVH) may lead to global pump failure. Recently, several endogenous molecular inhibitors of hypertrophy have been identified. Using real-time PCR, we compared the myocardial mRNA expression of these inhibitors in pressure-overload induced cLVH (severe aortic stenosis) and in volume overloadinduced eLVH (severe mitral regurgitation) in patients, and during the progression from cLVH to eLVH in pressure overload in rat. Each of these genes showed a unique temporal expression profile. Strikingly, except for SOCS-3, changes in gene expression of these negative regulators in rat cLVH and eLVH vs sham were recapitulated in human cLVH and eLVH. In particular, VDUP-1 and MCIP-1 were high in cLVH but expression levels were normal in eLVH, both in rat and human. These data indicate that during the progression of LVH, both in pressure and volume overload, expression levels of endogenous inhibitors of hypertrophy are modified and that these changes may have pathophysiological significance. In particular, MCIP-1 (the endogenous calcineurin inhibitor) and VDUP-1 (the endogenous inhibitor of thioredoxin) are potential molecular switches in the progression of LV hypertrophy.
Modulation of aminopeptidase N (APN) activity in the digestive tract by various factors would be important to adjust digestive and absorptive processes under different physiological and (or) environmental conditions. We studied the postprandial responses at different salinities and the effect of abscisic acid (ABA) and extracellular Ca2+ on APN activity in the hepatopancreas (the main site for nutrient digestion and absorption) of the model species Neohelice granulata (Dana, 1851). Enzyme activity was determined at different times (0, 24, 48, and 72 h) after feeding in crabs acclimated either to 35 psu (osmoconformation) or 10 psu (hyper-regulation). APN activity increased around 50% at 24 h after feeding at 35 psu, whereas no changes occurred at 10 psu. Enzyme activity was also assayed in the presence of ABA (1 × 10–4 mol·L–1) or extracellular Ca2+ (1 × 10–4 mol·L–1), showing increments of 60% and 56%, respectively. The results suggest a role of APN in postprandial adjustments and its modulation by different chemical messengers by direct effect on the hepatopancreas. Moreover, to our knowledge, this work is the first to show the effect of ABA on a digestive enzyme in the digestive tract of an animal.
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