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The relevance of the insulin-like growth factor-1 (IGF-1) system in several physiological processes is well-known in vertebrates, although little information about their temporal organization is available. This work aims to investigate the possible rhythmicity of the different components of the IGF-1 system (igf-1, the igf1ra and igf1rb receptors and the paralogs of its binding proteins IGFBP1 and IGFBP2) in the liver of goldfish. In addition, we also study the influence of two environmental cues, the light/dark cycle and feeding time, as zeitgebers. The hepatic igf-1 expression showed a significant daily rhythm with the acrophase prior to feeding time, which seems to be strongly dependent on both zeitgebers. Only igfbp1a-b and igfbp1b-b paralogs exhibited a robust daily rhythm of expression in the liver that persists in fish held under constant darkness or randomly fed. The hepatic expression of the two receptor subtypes did not show daily rhythms in any of the experimental conditions. Altogether these results point to the igf-1, igfbp1a-b, and igfbp1b-b as clock-controlled genes, supporting their role as putative rhythmic outputs of the hepatic oscillator, and highlight the relevance of mealtime as an external cue for the 24-h rhythmic expression of the IGF-1 system in fish., Ministerio de Ciencia e Innovación (España), Universidad Complutense de Madrid, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Europe calls for the end to fisheries discards, which means bringing all caught fish (subject to minimum sizes or quotas) to land. This decision is beneficial to the ecosystem, since it forces the selectivity of the fishing gears to improve. However, artisanal fishermen find themselves in a vulnerable situation where their subsistence depends on catches with small profit margins. An exemption to this landing obligation exists, as it is also ruled that those animals whose survival is scientifically guaranteed may be returned to the sea. Here we study the survival of Plectorhinchus mediterraneus captured by hookline and gillnet, as well as their physiological recovery. Survival exceeds 93% in both cases. The physiological assessment of primary (cortisol) and secondary (energy mobilization, acid-base and hydromineral balance, and immune system) stress responses indicates that surviving animals are able to recover after fishing. Thus, we propose the optimal size of capture of this species to achieve greater economic benefit. For this, we rely on the prices according to size in recent years, as well as on the growth curves of the species. In this way, by releasing fish of less than 1 kg, the current benefits could be multiplied between 2.3 and 9.6 times. This pilot study lays the groundwork for regulating artisanal fisheries through scientific data related to survival of discards along with information on the sale prices., Ministerio para la Transición Ecológica y el Reto Demográfico (España), Fundación Biodiversidad, European Commission, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Nocturnin (NOC) is a unique deadenylase with robust rhythmic expression involved in the regulation of metabolic processes in mammals. Currently, the possible presence of NOC in fish is unknown. This report aimed to identify NOC in a fish model, the goldfish (Carassius auratus), and to study the possible regulation of its expression by feeding. Two partial-length cDNAs of 293 and 223 bp, named nocturnin-a (noc-a) and nocturnin-b (noc-b), were identified and found to be highly conserved among vertebrates. Both mRNAs show a similar widespread distribution in central and peripheral tissues, with higher levels detected for noc-a compared with noc-b. The periprandial expression profile revealed that noc-a mRNAs rise sharply after a meal in hypothalamus, intestinal bulb, and liver, whereas almost no changes were observed for noc-b. Food deprivation was found to exert opposite effects on the expression of both NOCs (generally inhibitory for noc-a, and stimulatory for noc-b) in the three mentioned tissues. A single meal after a 48-h food deprivation period reversed (totally or partially) the fasting-induced decreases in noc-a transcripts in all studied tissues and the increases in noc-b expression in the intestinal bulb. Together, this study offers the first report of NOC in fish and shows a high dependence of its expression on feeding and nutritional status. The differential responses to feeding of the two NOCs raise the possibility that they might be underlying different physiological mechanisms (e.g., food intake, lipid mobilization, energy homeostasis) in fish., Ministerio de Economía, Industria y Competitividad (MINECO), Ministerio de Educación y Ciencia (MEC), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

La ingesta está sometida a un control complejo en el que intervienen numerososreguladores neurales, endocrinos y neuroendocrinos. Entre ellos, la ghrelina, sintetizadaprincipalmente por el estómago, o su equivalente en especies que carecen de él, destaca portratarse de la única hormona periférica de carácter orexigénico descrita hasta el momentoactual. La forma activa de esta hormona requiere la unión de un ácido graso de cadenamedia a la posición 3 de la cadena peptídica que es catalizada por la enzima ghrelina Oaciltransferasa(GOAT), localizada en la membrana del retículo endoplásmico. La acilaciónde la ghrelina es esencial para la unión a su principal receptor, el receptor de secretagogos dela hormona de crecimiento (GHS-R), lo que confiere a la GOAT un papel fundamental parael funcionamiento del sistema ghrelinérgico. En la actualidad, numerosos aspectosestructurales y funcionales de esta enzima permanecen desconocidos, especialmente enpeces.La síntesis y secreción de ghrelina, así como el funcionamiento del sistema ghrelinérgicoen general, están reguladas por diversos factores de naturaleza exógena, como laalimentación, y endógena, incluyendo un control hormonal y un control neural. Laalimentación es el principal regulador del sistema ghrelinérgico en los mamíferos, en los quela ghrelina se considera una señal iniciadora de la alimentación, ya que sus nivelescirculantes se elevan preprandialmente y disminuyen después de una comida. Además, laexpresión y secreción de esta hormona se ven incrementadas tras periodos de privación dealimento de distinta duración, y son reguladas por otros aspectos relacionados con la ingesta,como el patrón de alimentación y la composición de la dieta..., Food intake is subject to a complex control that involves numerous neural, endocrineand neuroendocrine regulators. Among them, ghrelin, synthesized mainly by stomach, or itsequivalent in stomachless species, is particularly important for being so far the onlyperipherally-produced peptide exerting a stimulatory effect on food intake. The active formof this hormone requires the attachment of a medium-chain fatty acid to the third serineresidue of the peptide, which is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT),located in the membrane of the endoplasmic reticulum. The acylation of ghrelin is essentialfor the peptide to bind to its main receptor, the growth hormone secretagogue receptor(GHS-R), conferring GOAT a key role in the functioning of the ghrelinrgic system.Currently, many structural and functional aspects of this enzyme remain unknown,especially in fish.The synthesis and secretion of ghrelin, as well as the functioning of the ghrelinergicsystem in general, are regulated by several exogenous factors, such as feeding, andendogenous factors, including a hormonal and a neural control. Feeding is the mainregulator of the ghrelinergic system in mammals, in which ghrelin is considered to be a mealinitiating signal, since its circulating levels increase preprandially and decrease after a meal.In addition, the expression and secretion of this hormone are increased after periods of fooddeprivation, and are regulated by other aspects related to food intake, such as the feedingpattern and the diet composition. In fish, the possible regulation exerted by feeding on thesynthesis and release of ghrelin has been scarcely investigated, and the few available studiesshow opposite observations in some cases, as occur with the effects of fasting...

Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish., Ministerio de Economía y Competitividad (MINECO), Ministerio de Educación y Ciencia (MEC), Natural Sciences and Engineering Research Council (NSERC) of Canada, Saskatchewan Health Research Foundation (SHRF) (Canada), CIHR, Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) (Argentina), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The circadian system is responsible for the temporal organisation of physiological functions which, in part, involves daily cycles of hormonal activity. In this review, we analyse the interplay between the circadian and endocrine systems in fishes. We first describe the current model of fish circadian system organisation and the basis of the molecular clockwork that enables different tissues to act as internal pacemakers. This system consists of a net of central and peripherally located oscillators and can be synchronised by the light–darkness and feeding–fasting cycles. We then focus on two central neuroendocrine transducers (melatonin and orexin) and three peripheral hormones (leptin, ghrelin and cortisol), which are involved in the synchronisation of the circadian system in mammals and/or energy status signalling. We review the role of each of these as overt rhythms (i.e. outputs of the circadian system) and, for the first time, as key internal temporal messengers that act as inputs for other endogenous oscillators. Based on acute changes in clock gene expression, we describe the currently accepted model of endogenous oscillator entrainment by the light–darkness cycle and propose a new model for non-photic (endocrine) entrainment, highlighting the importance of the bidirectional cross-talking between the endocrine and circadian systems in fishes. The flexibility of the fish circadian system combined with the absence of a master clock makes these vertebrates a very attractive model for studying communication among oscillators to drive functionally coordinated outputs., Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Ghrelin is an important gut-derived hormone with an appetite stimulatory role, while most of the intestinal hormones, including cholecystokinin (CCK), peptide YY (PYY) and glucagon-like peptide-1 (GLP-1), are appetite-inhibitors. Whether these important peptides with opposing roles on food intake interact to regulate energy balance in fish is currently unknown. The aim of this study was to characterize the putative crosstalk between ghrelin and CCK, PYY and GLP-1 in goldfish (Carassius auratus). We first determined the localization of CCK, PYY and GLP-1 in relation to ghrelin and its main receptor GHS-R1a (growth hormone secretagogue 1a) in the goldfish intestine by immunohistochemistry. Colocalization of ghrelin/GHS-R1a and CCK/PYY/GLP-1 was found primarily in the luminal border of the intestinal mucosa. In an intestinal explant culture, a significant decrease in prepro-cck, prepro-pyy and proglucagon transcript levels was observed after 60 min of incubation with ghrelin, which was abolished by preincubation with the GHS-R1a ghrelin receptor antagonist [D-Lys3]-GHRP-6 (except for proglucagon). The protein expression of PYY and GLP-1 was also downregulated by ghrelin. Finally, intraperitoneal co-administration of CCK, PYY or GLP-1 with ghrelin results in no modification of food intake in goldfish. Overall, results of the present study show for the first time in fish that ghrelin exerts repressive effects on enteric anorexigens. It is likely that these interactions mediate the stimulatory effects of ghrelin on feeding and metabolism in fish., Ministerio de Economía y Competitividad (MINECO), Natural Sciences and Engineering Research Council (NSERC) of Canada, Canada Foundation for Innovation to S. Unniappan, Saskatchewan Health Research Foundation (SHRF), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Ghrelin, a multifunctional gut-brain hormone, is involved in the regulation of gastric functions in mammals. This study aimed to determine whether ghrelin modulates digestive enzymes in goldfish (Carassius auratus). Immunofluorescence microscopy found colocalization of ghrelin, GHS-R1a and the digestive enzymes sucrase-isomaltase, aminopeptidase A, trypsin and lipoprotein lipase in intestinal and hepatopancreatic cells. In vitro ghrelin treatment in intestinal and hepatopancreas explant culture led to a concentration- and time-dependent modulation (mainly stimulatory) of most of the digestive enzymes tested. The ghrelin-induced upregulations of digestive enzyme expression were all abolished by preincubation with the GHS-R1a ghrelin receptor antagonist [D-Lys3]-GHRP-6, and most of them by the phospholipase C inhibitor U73122 or the protein kinase A inhibitor H89. This indicates that ghrelin effects on digestive enzymes are mediated by GHS-R1a, partly by triggering the PLC/PKC and AC/PKA intracellular signaling pathways. These data suggest a role for ghrelin on digestive processes in fish., Ministerio de Economía y Competitividad (MINECO), Ministerio de Educación y Ciencia (MEC), Natural Sciences and Engineering Research Council (NSERC) (Canada), Canada Foundation for Innovation, Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), (Argentina), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin’s physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish., Ministerio de Economía y Competitividad (MINECO), Natural Sciences and Engineering Research Council (NSERC) of Canada, Saskatchewan Health Research Foundation (SHRF), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

La ingesta está sometida a un control complejo en el que intervienen numerososreguladores neurales, endocrinos y neuroendocrinos. Entre ellos, la ghrelina, sintetizadaprincipalmente por el estómago, o su equivalente en especies que carecen de él, destaca portratarse de la única hormona periférica de carácter orexigénico descrita hasta el momentoactual. La forma activa de esta hormona requiere la unión de un ácido graso de cadenamedia a la posición 3 de la cadena peptídica que es catalizada por la enzima ghrelina Oaciltransferasa(GOAT), localizada en la membrana del retículo endoplásmico. La acilaciónde la ghrelina es esencial para la unión a su principal receptor, el receptor de secretagogos dela hormona de crecimiento (GHS-R), lo que confiere a la GOAT un papel fundamental parael funcionamiento del sistema ghrelinérgico. En la actualidad, numerosos aspectosestructurales y funcionales de esta enzima permanecen desconocidos, especialmente enpeces.La síntesis y secreción de ghrelina, así como el funcionamiento del sistema ghrelinérgicoen general, están reguladas por diversos factores de naturaleza exógena, como laalimentación, y endógena, incluyendo un control hormonal y un control neural. Laalimentación es el principal regulador del sistema ghrelinérgico en los mamíferos, en los quela ghrelina se considera una señal iniciadora de la alimentación, ya que sus nivelescirculantes se elevan preprandialmente y disminuyen después de una comida. Además, laexpresión y secreción de esta hormona se ven incrementadas tras periodos de privación dealimento de distinta duración, y son reguladas por otros aspectos relacionados con la ingesta,como el patrón de alimentación y la composición de la dieta...

The macronutrient composition of diets is a very important factor in the regulation of body weight and metabolism. Several lines of research in mammals have shown that macronutrients differentially regulate metabolic hormones, including ghrelin and nesfatin-1 that have opposing effects on energy balance. This study aimed to determine whether macronutrients modulate the expression of ghrelin and the nucleobindin-2 (NUCB2) encoded nesfatin-1 in goldfish (Carassius auratus). Fish were fed once daily on control, high-carbohydrate, high-protein, high-fat and very high-fat diets for 7 (short-term) or 28 (long-term) days. The expression of preproghrelin, ghrelin O-acyl transferase (goat), growth hormone secretagogue receptor 1 (ghs-r1) and nucb2/nesfatin-1 mRNAs was quantified in the hypothalamus, pituitary, gut and liver. Short-term feeding with fat-enriched diets significantly increased nucb2 mRNA levels in hypothalamus and liver, preproghrelin, goat and ghs-r1 expression in pituitary, and ghs-r1 expression in gut. Fish fed on a high-protein diet exhibited a significant reduction in preproghrelin and ghs-r1 mRNAs in the liver. After long-term feeding, fish fed on high-carbohydrate and very high-fat diets had significantly increased preproghrelin, goat and ghs-r1 expression in pituitary. Feeding on a high-carbohydrate diet also upregulated goat and ghs-r1 transcripts in gut, while feeding on a high-fat diet elicited the same effect only for ghs-r1 in liver. Nucb2 expression increased in pituitary, while it decreased in gut after long-term feeding of a high-protein diet. Collectively, these results show for the first time in fish that macronutrients differentially regulate the expression of ghrelinergic and NUCB2/nesfatin-1 systems in central and peripheral tissues of goldfish., Ministerio de Economía y Competitividad (MINECO), Natural Sciences and Engineering Research Council (NSERC) of Canada, Saskatchewan Health Research Foundation (SHRF), Canada Foundation for Innovation to Suraj Unniappan, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

The periprandial profile and effects of short- (7 days) and long-term (30 days) fasting on the ghrelinergic system were studied in goldfish (Carassius auratus). Plasma levels of acyl-ghrelin, desacyl-ghrelin, and ghrelin O-acyl transferase (GOAT) were analyzed by enzymoimmunoassays, and expression of preproghrelin, goat and growth hormone secretagogue receptors (ghs-r) was quantified by real-time PCR. Circulating levels of acyl-ghrelin and GOAT rise preprandially, supporting the role of acyl-ghrelin as a meal initiator in this teleost. Consistently, preproghrelin and ghs-r1a1 expression increases 1 h before feeding time in intestinal bulb, suggesting that this receptor subtype might be involved in the preprandial action of ghrelin in this tissue. Significant postfeeding variations are detected for preproghrelin in telencephalon, goat in telencephalon and hypothalamus, ghs-r1a1 in vagal lobe, ghs-r1a2 and ghs-r2a1 in hypothalamus and ghs-r2a2 in telencephalon and vagal lobe, especially in unfed fish. Short- and long-term fasting significantly increase preproghrelin expression in telencephalon and gut. Goat expression is upregulated by short-term fasting in telencephalon and hypothalamus, and by both short- and long-term fasting in gut. Expression of ghs-r increases by fasting in telencephalon, while an upregulation of type 2, but not type 1, receptors is observed in vagal lobe. In intestinal bulb, ghs-r1a2 transcripts increase after both short- and long-term fasting. These results show a high dependence of the ghrelinergic system on feeding and nutritional status in fish, and demonstrate for the first time a differential implication of the various components of this system suggesting different roles for the four ghrelinergic receptor subtypes., Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Ghrelin O-acyltransferase (GOAT) is the enzyme responsible for acylation of ghrelin, a gut-brain hormone with important roles in many physiological functions in vertebrates. Many aspects of GOAT remain to be elucidated, especially in fish, and particularly its anatomical distribution within the different brain areas has never been reported to date. The present study aimed to characterize the brain mapping of GOAT using RT-qPCR and immunohistochemistry in a teleost, the goldfish (Carassius auratus). Results show that goat transcripts are expressed in different brain areas of the goldfish, with the highest levels in the vagal lobe. Using immunohistochemistry, we also report the presence of GOAT immunoreactive cells in different encephalic areas, including the telencephalon, some hypothalamic nuclei, pineal gland, optic tectum and cerebellum, although they are especially abundant in the hindbrain. Particularly, an important signal is observed in the vagal lobe and some fiber tracts of the brainstem, such as the medial longitudinal fasciculus, Mauthneri fasciculus, secondary gustatory tract and spinothalamic tract. Most of the forebrain areas where GOAT is detected, particularly the hypothalamic nuclei, also express the ghs-r1a ghrelin receptor and other appetite-regulating hormones (e.g., orexin and NPY), supporting the role of ghrelin as a modulator of food intake and energy balance in fish. Present results are the first report on the presence of GOAT in the brain using imaging techniques. The high presence of GOAT in the hindbrain is a novelty, and point to possible new functions for the ghrelinergic system in fish., Ministerio de Economía y Competitividad (MINECO), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Cholecystokinin (CCK) plays a key role in the digestive physiology of vertebrates. However, very little is known about the role of CCK on intestinal functions in fish. The present study identifies two CCK receptor subtypes in a stomachless teleost, the goldfish (Carassius auratus), and investigates by using an in vitro system their involvement mediating the effects of the sulfated octapeptide of CCK (CCK-8S) on the motility of isolated proximal intestine. Partial-length mRNAs encoding two CCK receptor isoforms (CCKAR and CCKBR.I) were sequenced and the structural analysis showed that both receptors belong to the G-protein coupled receptor superfamily. Both gold- fish CCK receptor sequences were more closely related to zebrafish sequences, sharing the lowest similarities with cavefish and tilapia. The highest expression of goldfish CCKAR was observed along the whole intestine whereas the CCKBR gen was predominantly expressed in the hypothalamus, vagal lobe and posterior intestine. Application of CCK-8S to the organ bath evoked a concentration-dependent contractile response in intestine strips. The contractions were not blocked by either tetrodotoxin or atropine, suggesting that CCK-8S acts on the gut smooth muscle directly. Preincubations of intestine strips with devazepide and L365,260 (CCKAR and CCKBR receptor selective antagonists) showed that the CCK-8S-induced contraction could be partially mediated by the CCKAR receptor subtype, which is also the most abundant CCK receptor found in gastrointestinal tissues. In conclusion, two CCK receptors with a differential distribution pattern has been identified in goldfish, and the CCKAR subtype is mainly involved in the regulation of intestinal motility by the CCK-8S., Ministerio de Economía y Competitividad (España), Ministerio de Asuntos Exteriores y de Cooperación(España)-AECID, Ministerio de Educación Cultura y Deporte (España), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

L. Nisembaum and A.B. Tinoco are predoctoral fellowship from the Spanish MAEC-AECID and MICINN, respectively., Melatonin has been found in the digestive tract of many vertebrates. However, the enzymatic activity of the arylalkylamine-N-acetyltransferase (AANAT) and the hydroxindole-O-methyltransferase (HIOMT), the last two enzymes of melatonin biosynthesis, have been only measured in rat liver. Therefore, the first objective of the present study is to investigate the functionality of these enzymes in the liver and gut of goldfish, analyzing its possible daily changes and comparing its catalytic properties with those from the retina isoforms. The daily rhythms with nocturnal acrophases in retinal AANAT and HIOMT activities support their role in melatonin biosynthesis. In foregut AANAT activity also show a daily rhythm while in liver and hindgut significant but not rhythmic levels of AANAT activity are found. HIOMT activity is not detected in any of these peripheral tissues suggesting an alternative role for AANAT besides melatonin synthesis. The failure to detect functional HIOMT activity in both, liver and gut, led us to investigate other physiological substrates for the AANAT, as dopamine, searching alternative roles for this enzyme in the goldfish gut. Dopamine competes with tryptamine and inhibits retinal, intestinal and hepatic N-acetyltryptamine production, suggesting that the active isoform in gut is AANAT1. Besides, gut and liver produces N-acetyldopamine in presence of acetyl coenzyme-A and dopamine. This production is not abolished by the presence of folic acid (arylamine N-acetyltransferase inhibitor) in any studied tissue, but a total inhibition occurs in the presence of CoA-S-N-acetyltryptamine (AANAT inhibitor) in liver. Therefore, AANAT1 seems to be an important enzyme in the regulation of dopamine and N-acetyldopamine content in liver. Finally, for the first time in fish we found that dopamine, but not N-acetyldopamine, regulates the gut motility, underlying the broad physiological role of AANAT in the gut., Ministerio de Ciencia e Innovación (España), Universidad Complutense de Madrid//Banco de Santander, Ministerio de Asuntos Exteriores, Unión Europea y Cooperación (España), Agencia Española de Cooperación Internacional para el Desarrollo, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

La inyección ocular de ácido Kaínico es un método adecuado para la obtención de retinas de Xenopus laevis enriquecidas en fotorreceptores, siendo estas preparaciones un buen modelo para el estudio de la síntesis de melatonina en la retina. En los fotorreceptores de Xenopus laevis, la actividad TPH muestra un ritmo diario paralelo a los ritmos de actividad NAT y melatonina ocular. El ciclo luz-oscuridad dirige los principales sistemas de comunicación intracelular de los fotorreceptores de Xenopus laevis. La entrada de calcio a través de los canales tipo L desencadena una serie de procesos dependientes del complejo calcio-calmodulina, como una estimulación de la CaMKII y de la adenilato ciclasa tipo I. CaMKII fosforina directamente la TPH, activándola. La activación de la adenilato ciclasa aumenta el contenido de cAMP e induce una fosforilación de la TPH por la PKA. La dopamina a través de receptores D2 regula negativamente la actividad TPH de los fotorreceptores, mimetizando el efecto de la luz. Los niveles extracelulares de la adenosina regulan la síntesis de melatonina activando a través de receptores A2 las actividades TPH y NAT y mimetizando el efecto de la oscuridad