Maria Gabriella Denaro

Response to short term starvation of growth, haematological, biochemical and non-specific immune parameters in European sea bass (Dicentrarchus labrax) and blackspot sea bream (Pagellus bogaraveo)

Growth, haematological (haematocrit), biochemical (serum cortisol and glucose), and non-specific immune (lysozyme, serum haemolytic and haemagglutinating activities, extracellular respiratory burst activity) parameters, were monitored in European sea bass Dicentrarchus labrax and blackspot sea bream Pagellus bogaraveo subjected to a 31 days starvation compared to fed fish, to assess the responses to feed deprivation of these health status indicators. While haematocrit, serum cortisol, glucose and haemolytic activity of both species did not undergo significant variation following starvation, probably due to the short period applied, some non-specific immune parameters were affected significantly. In the starved sea bass, mucus lysozyme content doubled (1.8 U/mL) compared to the initial value. Haemagglutinating activity was significantly lower in starved sea bass than in fed fish after 31 days. In blackspot sea bream, a slight, not significant, reduction in haemagglutinating activity occurred 11 days after starvation. Respiratory burst activity decreased significantly in the starved fish. In spite of the limited number of examined parameters, the opportunity to use a panel of several indicators to obtain a more complete picture of health status in fish was underlined.

Short fasting and refeeding in red porgy (Pagrus pagrus, Linnaeus 1758): Response of some haematological, biochemical and non specific immune parameters

A short fasting-refeeding experience was applied to specimens of red porgy, Pagrus pagrus (Teleostei, Sparidae) to assess its effects on some physiological parameters. Haematological (haematocrit), biochemical (serum cortisol and glucose) and immunological (lysozyme, haemolytic and haemagglutinating activities) parameters were measured. For this study, two fish groups were considered: one was fasted for 14 days and then refed to satiation during further 7 and 15 days (indicated as fasted/refed group), the other was fed throughout the study and was taken as a control group. Significantly lower values were recorded for the condition index, the hepato-somatic index and viscero-somatic index in the fasted/refed group compared to the fed one. Fasting did not affect significantly the examined parameters, except for cortisol; refeeding for 7 days induced a significant increase in the haemoagglutinating titre and the spontaneous haemolytic activity, but when refeeding was extended to 14 days haemagglutinating and haemolytic values remained lower than those measured in fed fish.

Different effects of cGMP and cAMP in the intestine of the European eel, Anguilla anguilla

The regulation of salt absorption in the sea water cell intestine was studied by evaluating the effects of theophylline, 8 Br cyclic adenosine monophosphate, 8 Br cyclic guanosine monophosphate, atriopeptin III, porcine vasoactive intestinal peptide and prostaglandin E1 on the short-circuit current, the transepithelial voltage difference and conductance and on the dilution potentials. It was shown that theophylline increased the transepithelial conductance and reduced the magnitude of the dilution potentials, indicating that the drug increase the anion conductance of the tight junctions. In addition its inhibitory effect on short-circuit current and transepithelial voltage difference suggests that theophylline also affects the transcellular transport mechanisms. It was shown that 8 Br cyclic guanosine monophosphate and 8 Br cyclic adenosine monophosphate affect transcellular mechanisms underlying Cl− transport since both compounds reduced short-circuit current and transepithelial voltage difference; however, cyclic adenosine monophosphate is less effective since unlike cyclic guanosine monophosphate, even at maximal concentration, it was not able to completely abolish transepithelial voltage difference and short-circuit current. The effects of cyclic guanosine monophosphate and cyclic adenosine monophosphate were not additive even if cyclic guanosine monophosphate may produce further inhibition of ion transport in 8 Br cyclic adenosine monophosphate-treated tissues. In addition, cyclic guanosine monophosphate but not cyclic adenosine monophosphate reduced the magnitude of the dilution potentials, suggesting that cyclic guanosine monophosphate acts also on the paracellular pathway. Rat atriopeptin III, a peptide known to increase cyclic guanosine monophosphate cellular levels, behaved like 8 Br cyclic guanosine monophosphate since it lowered the dilution potentials and reduced short-circuit current and transepithelial voltage difference to near zero values, suggesting that the hormone modulates both paracellular and transcellular transport mechanisms, probably acting on the Na-K-2Cl cotransport. Agents acting via cyclic adenosine monophosphate, like porcine vasoactive intenstinal peptide and prostaglandin, behaved like 8 Br cyclic adenosine monophosphate. They were less effective in inhibiting ion transport and did not interfere with the paracellular pathway.

Requirement of HCO3- for Cl(-)-absorption in seawater-adapted eel intestine.

The role of HCO3−/CO2 buffer in Cl− absorption was examined in the in vitro perfused eel intestine adapted to seawater. Cl− absorption, expressed as short/circuit current (Isc), was measured in either 20 mM HCO3−/1% CO2 Ringer or HEPES Ringer, pH 8.0. Unilateral (mucosal or serosal) substitution of HCO3−/CO2 with HEPES/O2 was without effect on Isc and transepithelial voltage (Vt), whereas bilateral removal of HCO3−/CO2 reduced Isc and Vt by 50%, indicating that the presence of HCO3−/CO2 buffer at one side of the epithelium is sufficient to keep Cl− absorption at the maximum rate. We examined in further detail the individual components of the HCO3−/CO2 system that stimulates Cl− absorption. We found that, in tissues bathed with HEPES Ringer, addition of 1% CO2 to the luminal or serosal solution (final pH=7.6 in the chamber) had no effect on Isc and Vt, while both electrical parameters could be restored to control values by unilateral (luminal or serosal) substitution of HEPES Ringer with 20 mM HCO3−/1% CO2 Ringer or 20 mM HCO3−alone. Stimulation of Isc induced by unilateral (luminal or serosal) HCO3−/CO2 was inhibited by luminal or serosal 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS) (0,25 mM) or by serosal Na+ removal, whereas amiloride (1 mM), luminal or serosal, had no effect. Acetazolamide (0.1 mM, both sides) inhibited stimulation of Isc induced by luminal addition of HCO3−/CO2, whereas it was without effect when HCO3−/CO2 was added serosally or bilaterally. We reached the following conclusions, (a) Cl− absorption is stimulated by HCO3−/CO2 buffer via an increase in intracellular HCO3−concentration and/or pHi changes consequent to the HCO3−uptake mediated by HCO3−transport systems operating on both cell membranes, (b) A Na+-dependent SITS-inhibitable HCO3−transport mechanism operates at the basolateral membrane, (c) The transfer of HCO3−through the luminal membrane is mediated by the carbonic anhydrase enzyme located on the brush-border membranes of the enterocyte: the movement of HCO3−, via a SITS-sensitive transport system, occurs most likely in form of OH−, which originates from the dehydration reaction of HCO3−catalysed by the carbonic anhydrase. (d) There is no apparent amiloride-sensitive Na+/H+ antiporter on either cell membrane.

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla.

Abstract. The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(β-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.

H+ and Cl− secretion in the stomach of the teleost fish, Anguilla anguilla : stimulation by histamine and carbachol

Abstract The fundus of an eel stomach was mounted in an Ussing chamber and bathed with control Ringer on the serosal side and with unbuffered solution on the mucosal side. The gastric mucosa exhibited a mucosa negative transepithelial voltage (Vt), a “short circuit” current (ISC) and a small spontaneous acid secretion rate (JH). All these parameters were abolished by cimetidine treatment. Bilateral ion substitution experiments in tissues lacking spontaneous acid secretion suggested that a net Cl− transport from serosa to mucosa was responsible for the genesis of the ISC in the absence of H+ secretion. Serosal application of histamine (10−4 mol · l−1) or carbachol (10−4 mol · l−1) stimulated both ISC and JH. The action of carbachol was independent of histamine. The control as well as the histamine-stimulated ISC was sensitive to both serosal bumetanide (10−5 mol · l−1), inhibitor of the Na+-K+-2Cl− cotransport, and 4,4-diisothiocyano-stilbene-2,2-disulphonic acid (DIDS, 5 · 10−4 mol · l−1), inhibitor of the Cl−-HCO−3 exchange, while the ISC stimulated by carbachol was nullified by serosal DIDS. These data suggested that the non-acidic Cl− uptake across the serosal membrane was linked to the activity of both Na+-K+-2Cl− cotransport and Cl−-HCO−3 antiporter; histamine stimulated both transporters while carbachol was limited to the anion exchanger. The finding that the acid secretion was strictly dependent on serosal Cl− and was completely blocked by serosal DIDS suggested that the Cl− accompanying H+ secretion entered the cell through the serosal membrane by the Cl−-HCO−3 exchange. In addition, the acid secretion stimulated by carbachol was also dependent on serosal Na+ and sensitive to the application of 5-N-N-dimethyl-amiloride in the serosal bath, suggesting that the increased activity of the Cl−-HCO−3 during carbachol treatment was linked to the activation of serosal Na+-H+ exchange. The inhibitory effect of luminal omeprazole (10−4 mol · l−1) on acid secretion suggested the presence of the H+-K+ pump on the luminal membrane.

Protective effects of prostaglandins in the isolated gastric mucosa of the eel, Anguilla anguilla

Abstract The protective effect of endogenous prostaglandins on the fish gastric mucosa was evaluated by studying the effect of indomethacin and aspirin, known cyclooxigenase inhibitors, on the mucosal ulceration in the isolated gastric sacs of Anguilla anguilla. Gastric sacs devoid of muscle layers were incubated in the presence of indomethacin (10−4 mol · l−1) or aspirin (10−4 mol · l−1) in different experimental conditions. Both the anti-inflammatory drugs produced ulcers, but the effects were more severe in the presence of histamine and in the absence of HCO3− in the incubation bath. The effects of prostaglandin E2 (PGE2) on acid secretion rate (JH) and on alkaline secretion rate (JOH) were evaluated (with the aid of the pH stat method) in isolated gastric mucosa mounted in Ussing chambers. We found that PGE2 (10−8–10−5 mol · l−1) increased JH in a dose-dependent manner. In tissues pretreated with luminal omeprazole (10−4 mol · l−1), PGE2 stimulated gastric alkaline secretion. It was nullified by serosal removal of HCO3− or Na+ and by serosal ouabain (10−4 mol · l−1). These results suggested that prostaglandins also exert their protective effects in fish gastric mucosa. This protection seems partially due to a stimulation of exogenous HCO3− transport from the serosal to the mucosal side. It is likely that this transport is an active transcellular mechanism coupled to Na+ transport.

Temporal changes in digestive enzyme activities in the gastrointestinal tract of European eel (Anguilla anguilla) (Linneo 1758) following feeding

Changes occurring after feeding in the digestive enzyme activities of European eel were investigated to provide some insights into the digestive physiology of this fish. Total and specific proteases, amylase and lipase activities were measured using standard biochemical assays over a 24 h cycle in fed eels, compared to starved ones, under the same rearing conditions. In the gastrointestinal tract of fed eels quantitative changes started 4 h after feeding and continued later on; conversely, in starved eels enzyme activities remained unchanged over time. In fed eels, total and specific protease activities showed an overall increasing trend in the intestine, while in the stomach they progressively decreased to values 22–50% lower than those measured at the pre-feeding time; this behaviour probably reflected the progression of digesta along the intestinal tract. The prolonged secretory response of European eel to food ingestion proved its extended activity in the digestive process.

Effect of pH on transepithelial electrical parameters in seawater-adapted eel intestine

The sensitivity to external pH of Cl- absorption was studied in isolated stripped intestinal mucosa of the eel, Anguilla anguilla, mounted in Ussing chambers. Short-circuit current, transepithelial potential difference and conductance were measured in bathing solutions containing various combinations of HCO3--concentration (0–25 mmol·l-1), partial pressure of CO2 (0–76 mm Hg) and pH (6.9–7.9). A linear relationship was found between pH and short-circuit current in the range of pH studied both in HCO3-/CO2 Ringer and in Hepes Ringer. The pH effect was almost completely reversible. It was not affected by the presence of mucosal Ba2+ (10-3 mol·l-1) but it was inhibited by the presence of luminal (10-5 mol·l-1) or serosal (10-4 mol·l-1) bumetanide. The results obtained suggest that the Cl- absorption in the European eel intestine is pH sensitive. The data do not indicate whether the pH affects directly the Na+−K+−Cl- cotransport and/or the basolateral Cl- conductance or other mechanisms indirectly linked to Cl- absorption.

Effects of fasting and refeeding on the digestive tract of zebrafish (Danio rerio) fed with Spirulina (Arthrospira platensis), a high protein feed source

Abstract In the present work, morphological and molecular effects of short-term feed deprivation and refeeding with Spirulina (Arthrospira platensis) on zebrafish digestive tract were determined. Once elucidated the proximate composition of Spirulina feed, immunohistochemical and western blot analyses of peptide transporter (PepT1) and cholecystokinin (CCK8) were carried out in the gastrointestinal tract of zebrafish, previously morphologically investigated. Two and five fasting days caused not only morphostructural alterations, but also the downregulation of PepT1 and CCK8 proteins. Conversely, the recovery of normal morphological conditions, along with an increased PepT1 and CCK8 expression, were observed after refeeding with Spirulina. The increase of PepT1 expression in zebrafish may be responsible for the enhanced CCK8 secretion, so that both proteins may contribute to an improved digestion process during refeeding. These observations could be supported not only by compensatory mechanisms induced by fasting and refeeding but also by an higher protein quality of Spirulina-based diet.