The stress axis, stanniocalcin, and ion balance in rainbow trout.

In teleosts, the stress hormone cortisol and the calcium regulatory hormone stanniocalcin (STC) are both involved in the regulation of ion balance. Under stressful conditions, ion balance is easily disturbed as stressors via the stress signals they evoke disturb easily and primarily gill function. The gills are key in fish gas exchange and ion regulation. The present work evaluates the effect of the pivotal stress signal cortisol, the eventual output of the stress axis on STC secretion in freshwater rainbow trout (Oncorhynchus mykiss). Plasma cortisol levels were manipulated by intraperitoneal injections of porcine ACTH(1-39) or dexamethasone (Dex), and plasma cortisol, STC and mineral status were assessed. A perifusion assay of trout Stannius corpuscles was validated and used to study the direct effects of stress-related signals on STC release. In perifusion, cortisol, adrenocorticotropic hormone (ACTH), and dexamethasone did not affect STC release. ACTH injections increase plasma cortisol (corresponding to an acute stress) and STC concentrations, but did not affect mineral status. Dexamethasone injections resulted either in a classical hypocortisolinemia or, unexpectedly, in hypercortisolinemia. However, independently of the resulting cortisol status Dex induced a chronic stress effect, as indicated by decreased plasma Na, Cl, and Ca levels, and increased plasma STC concentrations. The increased STC secretion cannot be explained by the classical elevation of plasma calcium concentration. Thus, plasma parameters other than calcium could be involved and we propose that STC secretion might be stimulated also by a decrease of NaCl concentrations, implying a broader function than the classical hypocalcemic action of STC.

Composition of biomineral organic matrices with special emphasis on turbot (Psetta maxima) otolith and endolymph.

The soluble organic matrix (OM) of various biominerals (red coral skeleton, oyster shell, sea urchin test, turbot otolith, chicken eggshell) was extracted after demineralization with acetic acid. The protein content of the OM varies strongly from 0.02 to 1.6 microg/mg biomineral whereas proteoglycans present less variations (from 0.7 to 1.4 microg/mg biomineral). Electrophoresis of biominerals OM shows differences in their protein pattern although several bands are present in all matrices. OM of all biominerals shows carbonic anhydrase activity but no activity was detectable in the endolymph. OM of all biominerals also displays an anticalcifying activity. After separation of the OM extracts by chloroform-methanol, 80% of the anticalcifying activity was found in the methanol phase except in the urchin test. After OM precipitation with trichloracetic acid, 70% of the activities was found in the supernatants. Partial biochemical characterization suggests that the anticalcifying factor is a polyanionic and water-soluble molecule, which could be proteoglycans. The endolymph surrounding the otolith also displays an anticalcifying activity although its inhibitous activity was 50 times lower than that of the otolith OM. However, the anticalcifying activity of the endolymph is assumed by a proteic structure (80% activity precipitated with TCA treatment). Our results suggest that both carbonic anhydrase and anticalcifying activities are widespread and play a significant role in the regulation of biomineral formation. Results are discussed in relation to the calcification process that takes place at the fluid-mineral interface.

Daily variations of endolymph composition: relationship with the otolith calcification process in trout.

Ionic and organic parameters of the otolith calcification process in the trout Oncorhynchus mykiss were analysed in plasma and endolymph over the day:night cycle. Plasma pH remained constant and total CO(2) concentration was significantly lower (by 21%) during the day than at night. Calcifying parameters (total CO(2), total calcium concentration) were measured in the proximal and distal endolymphs and were unchanged in the latter during the day:night cycle, but fluctuated in the former. Non-collagenous protein and collagen concentrations in endolymph were higher (1.5- and 10-fold, respectively) during the day than at night. As there was no change in total calcium concentration, we propose that Ca(2+) increases during the dark period and was maximal by the end of the night when the total CO(2) concentration has also increased (by 14%). Measurements of endolymph pH in situ revealed significant differences between samples from proximal and distal endolymph (7.38 and 7.87, respectively), but no variation between values obtained during the day and at night. Thus, the saturation state of aragonite (Sa) in the proximal endolymph should fluctuate around unity during the day:night cycle, and CaCO(3) precipitation should occur when supersaturation is reached. The electrophoretic pattern of proximal endolymph showed variations in both major and minor components. Immunoblotting of endolymph, using a rabbit antiserum raised against the otolith soluble organic matrix revealed an increase in the expression of two proteins (65 kDa and 75 kDa) during the day period. We propose that organic matrix and calcium carbonate deposition on the otolith vary antiphasically: organic matrix deposition begins by the end of the day period, when the concentration of organic precursors is maximal in the endolymph, whereas CaCO(3) precipitation starts once the solubility of CaCO(3) is exceeded.

Otolith growth in trout Oncorhynchus mykiss: supply of Ca2+ and Sr2+ to the saccular endolymph.

Kinetic and pharmacological characteristics of Ca2+ fluxes across the saccular epithelium of trout were studied using a perfused isolated inner ear. 45Ca2+influx from the Ringer solution to the endolymph was 3-4 nmoles h(-1)microl(-1) endolymph, which corresponds to a global turnover rate of the endolymph calcium of 200 % h(-1). Ca2+ entry into the proximal endolymph was faster than into the distal fluid. Net Ca2+ movement across the saccular epithelium depended on the direction and intensity of the chemical gradient of calcium between the Ringer solution and the endolymph. Increasing the calcium concentration in the Ringer solution up to 4.4 mmol l(-1) provoked an accumulation of Ca2+ in both proximal and distal endolymphs, and equilibrium was reached about 30 min after the beginning of perfusion. Perfusion with calcium-free Ringer partially emptied the proximal compartment of calcium, whereas the calcium levels in the distal endolymph did not vary during 70 min of perfusion. Verapamil (10(-5) mol l(-1)) and cyanide (CN, 10(-3) mol l(-1)) did not modify the accumulation of Ca2+ within the endolymph in the presence of a favourable calcium chemical gradient. Furthermore the relationship between Ca2+ net fluxes and the chemical calcium gradient across the saccular epithelium was linear, indicating a passive diffusional mechanism via a paracellular pathway. Similar relationships were found for Sr2+ fluxes across the saccular epithelium in the presence of positive chemical gradients (1, 2 and 4 mmol l(-1) Sr2+). In vivo experiments in which trout were intraperitoneously injected with CaCl2 solution confirmed the tight relationship between the calcium levels in plasma and endolymph (both proximal and distal). Sampling proximal and distal endolymphs in trout and turbot saccules revealed a decreasing proximo-distal calcium gradient in endolymph of both fish species. The present results strongly suggest that the endolymph is supplied with Ca2+ and Sr2+ via a paracellular pathway located in the proximal area of the saccular epithelium.

Biochemical relationships between endolymph and otolith matrix in the trout (Oncorhynchus mykiss) and turbot (Psetta maxima).

This paper compares the organic compositions of the otolith and endolymph of trout and turbot. Irrespective of the method of demineralization (0.5 M EDTA or acetic acid), trout otoliths were found to be largely composed of proteins (48%), collagens (23%), and proteoglycans (29%). Collagen was only detectable in the EDTA-insoluble (0.30 microg/mg) and in the acetic acid-soluble fractions (0.53 microg/mg). The same compounds were found in the endolymph but in different proportions (proteins 85%, collagens 12%, and proteoglycans 3%). It was shown that the distribution of these compounds was not uniform within the endolymph. Proteins, collagens, and amino acids were 4, 10, and 3 times, respectively, more concentrated in the proximal (facing the macula) than the distal side whereas proteoglycans were 10 times more concentrated at the distal side. SDS PAGE analyses of proximal and distal samples of endolymph showed similar patterns suggesting that the spatial gradient of protein is quantitative and not qualitative. SDS PAGE comparison of endolymph and otolith samples showed only two proteins with similar molecular weights. We propose that collagen and protein gradients are involved in the organic matrix formation and otolith calcification process. Endolymphs from both trout and turbot display inhibitions of in vitro calcification although these inhibitions were 50 and 80 times, respectively, less than that of the otoliths. The inhibitory factor probably plays a significant role in the regulation of otolith calcification.

Chemical composition of saccular endolymph and otolith in fish inner ear: lack of spatial uniformity.

Fish otoliths provide a record of age, growth, and environmental influences. In both trout and turbot, spatial chemical investigation of the endolymph surrounding the otolith (sagitta) showed a lack of uniformity. Proteins, PO(3-)(4), and Mg(2+) were significantly more concentrated in the proximal (facing the macula) than distal zone, whereas the opposite was observed for K(+) and total CO(2) (totCO(2)). Na(+) concentration ([Na(+)]) was 20% higher in the proximal zone in trout but not in turbot. Total Ca and Cl(-) contents were uniformly distributed in both species. We propose that the endolymphatic gradients of protein and totCO(2) concentration within the endolymph are involved in the otolithic biocalcification process. Microchemical analyses of otolith sections by wavelength dispersive spectrometry showed a lack of spatial uniformity in the K/Ca and Na/Ca ratios, whereas the Sr/Ca ratio was uniform. There is a clear relationship between endolymph and otolith [K(+)], but the interpretation of the results for [Na(+)] needs further investigation. Thus the lack of uniformity in the otolith composition must be taken into account when investigating otolith microchemistry.

Ultrastructural study of the saccular epithelium of the inner ear of two teleosts, oncorhynchus mykiss and psetta maxima

The secretory cells and ionocytes of the saccular epithelium of the inner ear of trout (Oncorhynchus mykiss) and turbot (Psetta maxima) have been studied by electron microscopy. In these species, the saccular epithelium may be subdivided into four zones: the "macula", the "meshwork area", the "patches area", and the "intermediate area". In addition to the sensory "hair cells" and their supporting cells, the macula contains, at its periphery, "granular cells" that have the ultrastructural characteristics of secretory cells. The "meshwork area" around the macula contains large ionocytes endowed with pseudopods, many mitochondria, and three intracytoplasmic membrane systems (endoplasmic reticulum, tubular, and vesicular systems). The patches area, located at some distance from the macula, consists of groups of small mitochondria-rich ionocytes characterized by infoldings of their lateral plasma membrane. In the intermediate area, the size and organelle-content of cells decrease from the meshwork area to the patches area. There is no significant difference in cell composition or structure of the saccular epithelium between the trout and the turbot. The secreting cells might be involved in secretion of endolymph and formation of the otolith, whereas the ionocytes probably regulate the ionic composition of the endolymph.

Ionic composition of endolymph in teleosts: origin and importance of endolymph alkalinity.

Ionic (Na+, K+, Cl-, PO4(3-), pH), total CO2, total calcium and protein concentrations in the plasma and endolymph of the inner ear were compared in trout Oncorhynchus mykiss and turbot Scophthalmus maximus. In both species, saccular endolymph was characterized by high levels of K+ and total CO2 and in trout by an alkaline pH. The kinetic characteristics of proton secretion across the saccular epithelium of trout were investigation using a titration technique in which isolated saccules were mounted as closed sacs. The rate of proton secretion depends strongly on the pH of the Ringer's solution and secretion stops at a pH below 7.2. Proton secretion is driven by an energy-dependent mechanism involving basolateral ouabain-sensitive Na+/K+ exchangers. Proton secretion was partially inhibited by acetazolamide and completely inhibited in Na(+)-free Ringer or in the presence of 1 mmol l-1 amiloride. A cellular model stressing the importance of proton exchange through the saccular epithelium is proposed to explain the regulation of endolymph pH, a crucial factor for the deposition of otolith calcium.

Distribution of ionocytes in the saccular epithelium of the inner ear of two teleosts (Oncorhynchus mykiss and Scophthalmus maximus).

The saccular membranes of trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus) were examined to characterize specialized epithelial cells that might be responsible for ion exchange. The approach for localizing cell types was new for this tissue, as observations were made with a stereomicroscope and a light microscope in order to have a general view of the epithelium. No important differences between the two species were seen. The saccular tissue is a monolayer epithelium (except for the macula neural zone) surrounded by a layer of connective tissue invaded by many blood vessels. The use of the fluorescent probe DAPSMI and zinc iodide/osmium fixation-coloration defined two areas in which ionocytes were present. In the first, large ionocytes were grouped into a nearly complete, crowned meshwork around, but separated from, the macula. In the second area, opposite the macula, the ionocytes were smaller, cubical, and grouped in patches. Cells rich in Na+, K+-ATPase and carbonic anhydrase II were present in both areas. Contrary to previous studies in mammals and fish, ionocytes were also found in the epithelium of the saccule.

Simultaneous recognition of ionocytes and mucous cells in the gill epithelium of turbot and in the rat stomach.

A simple and rapid method is described which allows the recognition of different types of epithelial cells in one paraffin section. In the fish gill, the "chloride cells" (also called ionocytes or mitochondria-rich cells) appeared black and well contrasted after the zinc iodideosmium (ZIO) fixation, a contrast which is preserved after staining with alcian blue. All mucous cells appeared pale beige and can be clearly distinguished from the other cells but the known specificity of alcian blue for acid and sulfate mucins was lost. A complex tissue like the gill showed an interesting metachromasia. The combined ZIO fixation and alcian blue staining has also been applied on another epithelium, the rat stomach.

ELISA measurements of vasotocin and isotocin in plasma and pituitary of the rainbow trout: effect of salinity.

The fish neurohypophysial hormones arginine vasotocin (AVT) and isotocin (IT) were measured for the first time by ELISAs (in comparison with other techniques) in plasma and hypophysis of rainbow trout adapted stepwise from freshwater (FW) to seawater (SW). AVT concentrations were higher than IT in plasma and, conversely, lower in hypophysis. No difference appeared between FW and SW conditions, but plasma hormone concentrations fell when FW fish were moved to 1/3 SW and increased progressively when fish were moved from 1/3 SW to SW. Peptide values obtained in 1/3 SW may correspond to the lowest osmoregulatory constraints occurring in an isosmotic medium in comparison to FW or full SW. The data suggest that storage and/or release of AVT and IT differ, but vary in a similar way with external salinity, and that these peptides should play a role in teleost fish osmoregulation.

In vitro dose-response study of the effect of cadmium on eel (Anguilla anguilla) gill Na+/K(+)-ATPase activities.

The effect of cadmium exposure was studied in vitro on the ATPase activities of gill membrane microsomes from seawater- and freshwater-adapted eel (Anguilla anguilla) using a microassay technique. The basal activity (Mg(2+)-ATPase) was decreased by 40 and 25%, respectively, in seawater and freshwater preparations for the highest concentrations tested (respectively, 4 and 2 microM). The Na+/K(+)-ATPase activity was estimated either by potassium stimulation or by ouabain inhibition. This enzyme activity was inhibited by cadmium in a dose-dependent manner with a I50 of 146 +/- 9.3 nM. Neither the technique used to measure the enzyme nor the adaptative environment significantly changed the I50. The results are compared to results obtained in other groups and to the effects of cadmium on metal ion exchanges in fish.

Stanniocalcin kinetics in freshwater and seawater european eel (Anguilla anguilla).

The kinetics and hypocalcemic potency of stanniocalcin (STC) were examined in freshwater and seawater eels. The secretion rate and the metabolic clearance rate of STC were calculated from the STC disappearance curve after intra-arterial injection of trout STC. Basal plasma STC concentrations in freshwater and seawater eels did not differ but the STC secretion rate and metabolic clearance rate in seawater eel were 70-75% higher than in FW eel. The increased STC distribution space in seawater eels suggests that the STC receptor density was increased. STC had a higher hypocalcemic potency in seawater than in freshwater eels. These observations support the hypothesis that seawater fish require more hormonal control over transcellular influx of calcium than freshwater fish.

An enzyme-linked immunosorbent assay for stanniocalcin, a major hypocalcemic hormone in teleost.

An enzyme-linked immunosorbent assay (ELISA) was developed to quantify stanniocalcin (STC) levels in tissue extracts and plasma samples. The detection limit of the competitive ELISA described is 0.2 ng STC per well, allowing detection of 3.7 pmol.liter-1 (assuming a molecular mass of 54,000 Da for native STC). The particular antiserum detects STC in plasma obtained from a variety of freshwater and seawater species. In freshwater post-smolt Salmo salar plasma, STC levels were significantly lower (around 0.74 nmol.liter-1) than those of seawater smolts (around 2.78 nmol.liter-1). Seven days after removal of Stannius corpuscles from freshwater eels a significant hypercalcemia was observed as well as a drop in plasma STC levels (from 2.33 to 0.67 nmol.liter-1).

Effects of ambient ion concentrations on gill ATPases in fresh water eel,Anguilla anguilla.

Branchial activities of Na(+),K(+)-ATPase (ouabain sensitive), Mg(2+) ATPase (ouabain insensitive) and kinetic analysis of high and low affinity Ca(2+) ATPase were measured inAnguilla anguilla that had been acclimated to demineralized water (DW, Ca < 10 µM), freshwater (FW, Ca = 2 mM), and Low calcium freshwater (L-Ca, Ca = 0.9 mM). Na(+),K(+)-ATPase activity decreased while ouabain insensitive activity increased when ambient Ca(2+) decreased. Two kinetic forms of Ca(2+) ATPase could be resolved in each environmental condition. The stimulation coefficients of both sites or enzymes were not affected by ambient Ca(2+) concentrations. The maximal velocity of both the high and the low affinity Ca(2+) ATPase was increased when external Ca(2+) was decreased during acclimation. The low affinity Ca(2+) ATPase and the Mg(2+) stimulated enzyme could be a non specific enzyme accepting either Ca(2+) or Mg(2+). Results are compared with previous results in the literature and in relation to the branchial morphology and ionic exchanges in fish.

Ca2+-stimulated ATPase activities in the gill of the eel: interactions of Mg2+ ions.

Enriched plasma membrane preparations of the branchial epithelium of freshwater-adapted eels were used to study adenosine triphosphatase (ATPase) activities insensitive to ouabain and responding to Ca2+ and Mg2+. Ca2+ induced ATP hydrolysis; two kinetics were observed in the presence or absence of chelators, one with a high-affinity site (0.3 microM) and one with a lower affinity site (10-20 microM). The high-affinity Ca2+ site or enzyme had a prerequisite for Mg2+ (endogenous Mg2+ being sufficient to satisfy the Mg2+ need) but was inhibited by exogenous Mg2+ (Ki0.5 less than 10 microM Mg2+). The low-affinity site or enzyme appears to have kinetic parameters comparable to those found for Mg2+-induced ATP hydrolysis. In the absence of Ca2+ ligands and with no exogenous Mg2+, the two Ca2+ sites or enzymes can be considered stimulated. The results are discussed in relation to the branchial ion environment and transport ion capacities.

Effects of ovine prolactin on calcium uptake and distribution in Oreochromis mossambicus.

Ovine prolactin stimulated the net uptake rate of Ca2+ from the water by 96%, produced frank hypercalcemia, and increased total bone calcium content in fed rapidly growing freshwater male tilapia, Oreochromis mossambicus. It did not, however, alter the size of the readily exchangeable bone calcium pool. The increase in calcium accumulation resulted from an increase in whole-body Ca2+ influx and a decrease in Ca2+ efflux. It is concluded that prolactin exerts an important control over Ca2+ exchange between the fish and its environment and that through its hypercalcemic action prolactin indirectly facilitates bone mineralization.

Whole-body calcium flux rates in cichlid teleost fish Oreochromis mossambicus adapted to freshwater.

Radiotracer techniques were used to measure influx and efflux rates of Ca2+ in freshwater-adapted Oreochromis mossambicus. The influx rate of Ca2+ is related to body weight (W) as Fin = 50W0.805 nmol Ca2+/h. For a 20-g fish the calculated influx rate was 558 nmol Ca2+/h, and this was attributed largely to extraintestinal uptake since the drinking rate was estimated to be only 28 microliter water/h, which corresponds to an intake of 22.4 nmol Ca2+/h. The Ca2+ efflux rate was calculated using the initial rate of appearance of radiotracer in the ambient water and the specific activity of plasma Ca2+. Tracer efflux rates were constant over 6-8 h, which indicated that there was no substantial loss of tracer in either the urine or the feces because this would have resulted in random bursts of tracer loss. Efflux rates then primarily represent integumentary and presumably branchial efflux rates. The efflux rate of Ca2+ is related to body weight as Fout = 30W0.563 nmol Ca2+/h, which means an efflux rate of 162 nmol Ca2+/h for a 20-g fish. The net whole-body Ca2+ influx, calculated as Fnet = Fin - Fout, was 396 nmol/h for a 20-g fish, which proves that the ambient water is an important source of Ca2+.

The use of isolated fish opercular epithelium as a model tissue for studying intrinsic activities of loop diuretics.

Isolated opercular epithelia of killifish (Fundulus heteroclitus) were mounted in an Ussing chamber. The epithelia displayed a transepithelial electrical potential difference (PD) of 10.6 +/- 0.3 mV (sea-water side negative) and a short-circuit current (SCC) of 72.1 +/- 2.1 microA cm-2. The electrical resistance was 160 +/- 3 omega cm2 (mean +/- SE, n = 269). The unidirectional flux of 36Cl from blood side to sea-water side compared well with the SCC. No net flux of 22Na or 24Na across the epithelium was observed. Raising of cyclic AMP levels by theophylline, 3-isobutyl-I-methyl-xanthine, isoprenaline and forskolin, increased SCC and PD. Prostaglandins PGE2 and to some extent PGF2 alpha inhibited SCC and PD. Inhibition of Na+-K+-ATPase by ouabain and orthovanadate reduced SCC and PD. Pretreatment of the epithelium with the stilbene disulphonic acid (DIDS) did not prevent the action of orthovanadate. Different types of diuretics were tested, but only the loop diuretics bumetanide, piretanide, and furosemide, rapidly and strongly inhibited PD and SCC and unidirectional 36Cl serosal to mucosal flux. Dose-response curves for these agents were parallel and EC50 values for effects on SCC were 40, 52, and 295 microM, respectively. The same relative activities of these diuretics have been seen in the renal thick ascending limb of Henle's loop (TALH). It is concluded that the killifish opercular skin responds to hormonal stimuli and various pharmacological agents in a manner similar to that of mammalian renal TALH. It should therefore be a useful model for studies of the modes of action and the structure-activity relationships of diuretics which act by inhibition of chloride transport or Na+-K+-ATPase activity.

Estimation of sodium uptake through the gill of the rainbow trout Salmo gairdneri.

Sodium exchanges through the gill epithelia were estimated in the rainbow trout, Salmo gairdneri, using the perfused head technique. The head tissues accumulate radioactivity. In addition there exists an extrabranchial entry of sodium, which is 20 times smaller than the branchial one. The study of the evolution of venous and arterial sodium specific activities as a function of time is necessary before flux measurements are achieved. Contrary to previous studies, an uptake at the primary lamella level is demonstrated. Uptake of sodium through the secondary lamellae can be measured rapidly while at least 30 min are needed for measurement of uptake through the primary lamellae. Chloride cells of the primary lamellae contribute to about 20% of the total sodium uptake.