Periprandial changes and effects of short- and long-term fasting on ghrelin, GOAT, and ghrelin receptors in goldfish (Carassius auratus).

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.

Two cholecystokinin receptor subtypes are identified in goldfish, being the CCKAR involved in the regulation of intestinal motility.

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 goldfish 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.

The arylalkylamine-N-acetyltransferase (AANAT) acetylates dopamine in the digestive tract of goldfish: a role in intestinal motility.

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.

Balance of pro-apoptotic transforming growth factor-beta and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina.

Transforming growth factor (TGF)-beta and insulin display opposite effects in regulating programmed cell death during vertebrate retina development; the former induces apoptosis while the latter prevents it. In the present study we investigated coordinated actions of TGF-beta and insulin in an organotypic culture system of early postnatal mouse retina. Addition of exogenous TGF-beta resulted in a significant increase in cell death whereas exogenous insulin attenuated apoptosis and was capable of blocking TGF-beta-induced apoptosis. This effect appeared to be modulated via insulin-induced transcriptional down-regulation of TGF-beta receptor II levels. The analysis of downstream signalling molecules also revealed opposite effects of both factors; insulin provided survival signalling by increasing the level of anti-apoptotic Bcl-2 protein expression and phosphorylation and down-regulating caspase 3 activity whereas pro-apoptotic TGF-beta signalling reduced Bcl-2 mRNA levels and Bcl-2 phosphorylation and induced the expression of TGF-induced immediate-early gene (TIEG), a Krüppel-like zinc-finger transcription factor, mimicking TGF-beta activity.

Regulation of tryptophan hydroxylase activity in Xenopus laevis photoreceptor cells by cyclic AMP.

The aim of this study was to investigate the role of cyclic AMP in the regulation of tryptophan hydroxylase activity localized in retinal photoreceptor cells of Xenopus laevis, where the enzyme plays a key role in circadian melatonin biosynthesis. In photoreceptor-enriched retinas that lack serotonergic neurons, tryptophan hydroxylase activity is markedly stimulated by treatments that increase intracellular levels of cyclic AMP or activate cyclic AMP-dependent protein kinase, including forskolin, phosphodiesterase inhibitors, and cyclic AMP analogues. In contrast, cyclic AMP has no effect on tryptophan hydroxylase mRNA abundance. Experiments using cycloheximide and actinomycin D demonstrate that cyclic AMP exerts its regulatory effect via posttranslational mechanisms mediated by cyclic AMP-dependent protein kinase. The effect of cyclic AMP is independent of the phase of the photoperiod, suggesting that the nucleotide is not a mediator of the circadian rhythm of tryptophan hydroxylase. Cyclic AMP accumulation is higher in darkness than in light, as is tryptophan hydroxylase activity. Furthermore, the stimulatory effect of forskolin and that of darkness are inhibited by H89, an inhibitor of cyclic AMP-dependent protein kinase. In conclusion, cyclic AMP may mediate the acute effects of light and darkness on tryptophan hydroxylase activity of retinal photoreceptor cells.

Melatonin synthesis in the greenfrog retina in culture: I. Modulation by the light/dark cycle, forskolin and inhibitors of protein synthesis.

Melatonin is synthesized in the pineal gland and the retina of vertebrates. Retinal serotonin N-acetyltransferase (NAT) activity and melatonin show a daily rhythm with high levels during the dark phase of the photocycle. In some vertebrates, these retinal NAT and melatonin rhythms are maintained in vitro. The aim of present work is to develop an eyecup culture system for the greenfrog (Rana perezi), suitable to analyze the mechanisms of regulation of melatonin synthesis by simultaneous determination of NAT activity and melatonin release. The R. perezi eyecups released melatonin to the culture medium in a rhythmic manner at least over a 27-h period under photocycle conditions. NAT activity and melatonin rhythms were similar to that observed in vivo under natural environmental conditions. Rana perezi retina exhibits a pronounced photosensitivity in vitro. Forskolin increased up to 2-fold the NAT activity and 4-fold the melatonin production at any lighting conditions. The addition of the translation inhibitor, cycloheximide, to the medium reduced significantly both nocturnal NAT activity and melatonin release, suggesting that de novo protein synthesis is produced daily during darkness. Actinomycin D, a transcription inhibitor, needs a longer time of action, because pre-existing mRNA must be depleted before the inhibition of melatonin release can be observed. The eyecup culture system is highly sensitive to light and chemical factors, which makes it particularly suitable as a model for the neurochemical analysis of melatonin biosynthesis in the retina of Rana perezi.

Melatonin synthesis in the greenfrog retina in culture: II. Dopaminergic and adrenergic control.

Serotonin N-acetyltransferase (NAT) activity and melatonin show a daily rhythm with high levels at night. Although the rhythmic properties of NAT and melatonin are similar in pineal gland and retina, great differences in the light perception and transmission mechanisms exist. We have analyzed the effects of adrenergic and dopaminergic agents on greenfrog (Rana perezi) eyecup culture, in order to identify the receptors involved in the regulation of retinal melatonin synthesis. A D2-like receptor is directly involved in the regulation of NAT activity and melatonin release in R. perezi retina. Quinpirole mimics the effect of light, reducing the darkness-stimulated NAT activity and melatonin release, while sulpiride antagonized these actions. Neither D1-agonist (SKF 38393) nor D1-antagonist (SCH 23390) had effect on NAT activity. However, a significant inhibition of darkness-evoked melatonin release was produced by SKF 38393 after 6 hours of culture. The beta- and antagonist1-agonists showed a clear inhibition. However, a direct effect of beta, alpha1 and D1-agonists on photoreceptors is unproven, being more probable that the adrenergic actions imply a non-photoreceptor retinal cell. In conclusion, eyecup culture of Rana perezi revealed a dopaminergic control of melatonin synthesis and a possible modulation of dopaminergic tone by adrenergic receptors. Melatonin release is a more sensitive parameter than NAT activity to the action of neuroactive agents, suggesting that melatonin synthesis can be regulated by more than one enzymatic step in Rana perezi.

Diurnal rhythms of tryptophan hydroxylase activity in Xenopus laevis retina: opposing phases in photoreceptors and inner retinal neurons.

Tryptophan hydroxylase (TPH) is the first enzyme in the biosynthetic pathways of melatonin in photoreceptor cells and of serotonin in amacrine cells. To assess the regulation of TPH activity in photoreceptor cells, we pretreated retinas with kainic acid. The neurotoxin selectively killed inner retinal neurons while sparing photoreceptors. TPH activity in both control and kainate-treated retinas undergoes a day-night rhythm. The rhythms in both preparations fit sinusoidal functions. However, the rhythm in intact retinas peaks at midday while that in kainate-lesioned retinas does so at midnight. The daily rhythm of tryptophan hydroxylase activity in photoreceptors parallels that of melatonin release. Comparing the mean level of activity in rhythms of intact and lesioned retinas, we calculate that the TPH activity in photoreceptors represents 24% of the total activity. Therefore, the TPH activity measured in intact retinas reflects mainly the enzymatic activity in serotonergic neurons, masking that from photoreceptors. In contrast, the levels and diurnal variation of TPH mRNA did not differ in intact and kainate-lesioned retinas indicating that measurements of TPH mRNA content reflect primarily that in photoreceptor cells. Thus, TPH mRNA levels and enzyme activity are differentially regulated in amacrine neurons and photoreceptor cells. This differential regulation markedly impacts the patterns of daily rhythms observed in the intact retina.

Inhibitory effect of serotonin on feeding behavior in goldfish: involvement of CRF.

The possible action of 5-HT on feeding behavior in goldfish has been studied. Food intake was significantly reduced by intracerebroventricular (ICV) injection of serotonin (5-HT, 10 microg) at 2 h postinjection. After peripheral (intraperitoneal) administration of 5-HT (1 and 10 microg/g bw), no significant modifications in food intake were detected. Thus, it can be concluded that there is a central anoretic action of 5-HT in teleost fish. Taking in mind the inhibitory effect of corticotropin releasing factor (CRF) on feeding in teleosts and the interactions between 5-HT and CRF described in mammals, we investigated the possible involvement of CRF as mediator of the 5-HT anoretic action in goldfish. The ICV pretreatment with alpha-Helical CRF[9-41](20 microg) partially blocked the inhibitory effect of 5-HT on food consumption in goldfish. These results show that CRF mediates, at least in part, the 5-HT-induced feeding inhibition in goldfish. On the other hand, the alterations in hypothalamic indoleamines content evoked by ICV treatments would suggest that the activation of CRF neurons by 5-HT appears to inhibit hypothalamic serotoninergic transmission, supporting the intermediate role of this neuropeptide in the central anoretic effect of 5-HT in goldfish.

Effect of alpha-helical-CRF[9-41] on feeding in goldfish: involvement of cortisol and catecholamines.

The anoretic effect of corticotropin-releasing factor (CRF) was not dependent on adrenal activation in goldfish (Carassius auratus). Moreover, an interaction between CRF and the hypothalamic catecholaminergic system in the central regulation of food intake was observed. The intracerebroventricular (icv) administration of CRF increased cortisol levels and reduced food intake and hypothalamic norepinephrine and dopamine content at 2 hr postinjection, with these effects reversed by alpha-helical CRF[9-41] pretreatment. The anoretic effect of CRF was independent of the circulating cortisol increase, because it was only evoked after icv injections but not after intraperitoneal (ip) administration. Furthermore, the increase in plasma cortisol levels induced by ip administration of this steroid did not modify feeding.

Effect of constant and fluctuating temperature on daily melatonin production by eyecups from Rana perezi.

We analysed the effect of daily temperature cycles in relation to constant temperature on day/night melatonin synthesis in frog eyecups in culture. Eyecups were cultured for 24 h under 12L:12D photoperiod and two thermal regimes, constant temperature (25, 15 and 5 degrees C) and thermoperiod (WL/CD, thermophase coinciding with photophase and cryophase coinciding with scotophase; and CL/WD, cryophase coinciding with photophase and thermophase coinciding with scotophase). A negative correlation between ocular serotonin N-acetyltransferase activity and culture temperature for both diurnal and nocturnal activities has been observed. This effect of increased ocular activity at low temperature is more pronounced than the well-known stimulatory effect of darkness, and it does not depend on the photoperiod phase. The lack of interactions between the phase of photoperiod and culture temperature indicates that the effects of both factors are independent. Night-time temperature is the key factor in determining the amplitude of the melatonin rhythm in the Rana perezi retina. However, daytime temperature can not counteract the inhibitory effect of light on ocular melatonin synthesis.

Differential characteristics and regulation of arylamine and arylalkylamine N-acetyltransferases in the frog retina (Rana perezi).

Arylamine N-acetyltransferase activity (A-NAT: E.C.2.3.1.5) from Rana perezi retina was studied using p-phenetidine as specific substrate. Enzyme characteristics and regulation were compared with respect to the arylalkylamine N-acetyltransferase (AA-NAT: E.C.2.3.1.87) from the same tissue. A-NAT activity is distributed in both neural retina and choroid-pigmented epithelium complex, showing a 10-fold higher specific activity in neural retina. In contrast, AA-NAT activity is restricted to neural retina. Subcellular localization in neural retina indicated that both enzymatic activities are in the supernatant fraction (39,000 g, 20 min). p-Phenetidine acetylation was linear as a function of the neural retina amount in the assay (1/16 to 1 retina), and it is insensitive to phosphate buffer pH in the range 6.5-8.4. A-NAT kinetic showed a hyperbolic shape for both cosubstrates. Kinetic constants were KM = 11.2 microM, Vmax = 0.49 nmol/h/mg prot. for p-phenetidine (50 microM acetyl-CoA), and KM = 113.4 microM, Vmax = 3.1 nmol/h/mg prot. for acetyl-CoA (5 mM p-phenetidine). The additivity test for both enzymatic activities in retina homogenates demonstrated that both acceptor amines do not compete for the catalytic sites. Serotonin addition in the assay modifies differentially the kinetic characteristics of both enzymes. Serotonin acted as a strong mixed inhibitor, mainly competitive in nature (competitive Ki = 18.1 microM; non-competitive Ki = 1.9 mM) for AA-NAT. However, it acted as a weak inhibitor with respect to A-NAT, mainly non-competitive, (competitive Ki = 5.7 mM; non-competitive Ki = 8.7 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin N-acetyltransferase activity as a target for temperature in the regulation of melatonin production by frog retina.

The adaptive mechanisms of serotonin N-acetyltransferase (NAT) activity in the regulation of melatonin synthesis in frog retina in the face of chronic and acute temperature changes have been investigated. We performed thermal acclimation experiments to test different environmental temperatures at two seasons of the year (summer and winter), followed by the set-up of an eyecup culture system to investigate the acute effects of temperature on NAT activity and melatonin production daily rhythms. Low temperature induced a significant increase in NAT activity, independent of both the time of the photocycle (midday or midnight) and the season of the year (winter or summer). Acute cold-induced stimulation of NAT activity may be associated with lower decreases in the enzyme synthesis rate, rather than decreases in the degradation rate. In contrast, acclimation to warm temperature (25 degrees C) stimulated ocular melatonin production. Nocturnal melatonin production in eyecups cultured at 25 degrees C was significantly higher than in eyecups cultured at 5 degrees C. We suggest that this discrepancy in thermal regulation of melatonin synthesis can be justified by a seasonal variation in serotonin content within the photoreceptor cells, which determines the thermal response of melatonin production through changes in NAT kinetics.

Ontogeny of ocular serotonin N-acetyltransferase activity daily rhythm in four anuran species.

The profile of daily ocular serotonin N-acetyltransferase (NAT) rhythm has been examined during the development of four anuran amphibians in different environments and of varying phylogenetic origins: Xenopus laevis, Discoglossus pictus, Rana perezi, and Bufo calamita. A significant day/night rhythm of ocular NAT with the acrophase at midnight was first detected at stages 43/44 and 25 in X. laevis and R. perezi, respectively. Total enzymatic activity increased with body weight throughout development, although both mean levels and amplitudes of NAT activity per milligram of protein significantly decreased; this attentuation of rhythm was more significant in postmetamorphic froglets, and the acrophase was delayed about 1 hr. There were no significant differences in daily NAT profiles before and after metamorphosis in D. pictus and B. calamita. The amplitude of the NAT rhythm was maximal (> 10-fold nocturnal values) in Xenopus compared with other species (2- to 3-fold).

Serotonin N-acetyltransferase (NAT) activity and melatonin levels in the frog retina are not correlated during the seasonal cycle.

The seasonal pattern of daily serotonin N-acetyltransferase (NAT) and melatonin rhythms in the lateral eye of Rana perezi has been characterized to assess any seasonal relationship between enzymatic activity and melatonin production and to evaluate the photothermal responses of such rhythms. In winter there was no daily rhythm either in NAT activity or in melatonin production, although the highest values of NAT activity and lowest melatonin concentrations were measured at this time. Ocular melatonin showed a high-amplitude diurnal rhythm during the summer, whereas a low-amplitude rhythm was observed in spring and autumn. Diurnal melatonin levels were temperature-independent; however, nocturnal melatonin production significantly increased with temperature. Ocular NAT was always higher at lower than at higher temperatures (two- to threefold), both at night and during the day. As suggested for pineal melatonin production in other poikilotherm species, photoperiod controls the duration of the melatonin nocturnal rise, whereas temperature regulates the amplitude of the daily melatonin rhythm in the frog retina. The lack of correlation between daily NAT and melatonin rhythms in the frog retina may result from the high thermal sensitivity of the ocular NAT and a temperature-dependent inhibition substrate of NAT activity.