Endurance exercise modifies the circadian clock in zebrafish (Danio rerio) temperature independently.

AIM: Several rodent and human studies revealed that physical exercise acts as a non-photic zeitgeber for the circadian clock. The intrinsic entraining mechanism is still unknown, although it was assumed that the exercise-mediated increase in core temperature could be the underlying zeitgeber. As the homoeostatic control of mammalian core temperature interferes strongly with the investigation of this hypothesis, the present study used the poikilotherm zebrafish to answer this question. METHODS: Gene transcription levels of the two circadian core clock genes period1 and clock1 were quantified using real-time qPCR of whole animal zebrafish larvae. RESULTS: Long-term endurance exercise of zebrafish larvae aged 9-15 days post-fertilization (dpf) or 21-32 dpf at a constant water temperature of 25 °C caused significantly altered transcription levels of the circadian genes period1 and clock1. Cosinor analysis of diurnal transcription profiles obtained after 3 days of swim training revealed significant differences regarding acrophase, mesor and amplitude of period1, resulting in a phase delay of the gene oscillation. After termination of the exercise bout, at 15 dpf, oscillation amplitudes of both circadian genes were significantly reduced. CONCLUSION: The results showed that physical exercise is able to affect the transcription of circadian genes in developing zebrafish larvae. Considering the poikilothermy of zebrafish, an exercise-mediated change in body core temperature could be excluded as the underlying intrinsic zeitgeber. However, the day-active zebrafish arises as a useful model to address the synchronizing effect of exercise on the circadian clock.

Influence of swim training on cardiac activity, tissue capillarization, and mitochondrial density in muscle tissue of zebrafish larvae.

Larval zebrafish (Danio rerio) of two different age classes ("swim-up" larvae, 9 days old; "free-swimming" larvae, 21 days old) were exposed to either an endurance/continuous training or interval training. Control animals were kept in stagnant water. A comparison of cardiac activity of trained (either endurance or interval) and untrained animals at the end of the training regime revealed no differences in heart rate, end-diastolic and end-systolic ventricular volume, and cardiac output. Training also had no influence on the concentration of erythrocytes in the blood. Thus, at the level of total oxygen transport in the blood, training did not provoke any improvement during the first 32 days of development. Significant changes, however, were observed at the tissue level. In free-swimming larvae [i.e., between 21 and 32 days postfertilization (dpf)] endurance training increased the capillarization of both axial muscle caudal to the anus and the tail fin. In addition, mitochondrial density of red and intermediate muscle fibers increased significantly. In contrast to capillarization, even swim-up larvae, trained between 9 and 15 dpf, were affected. The observed increase in mitochondrial content indicates a high demand for oxygen and energy-rich metabolites for oxidative phosphorylation. In older larvae, this is met by the increase in capillarization that improves the blood supply and with it the required oxygen and metabolite supply of muscle tissue. Both of these adaptational changes result in a reduction of diffusion distances (between capillary and muscle fiber as well as mitochondria) and may contribute to a higher resistance toward oxygen deficiency. Furthermore, this study indicates that plasticity of muscle tissue is already established in early stages of development at both the tissue and cellular levels.

Expression of two isoforms of the vacuolar-type ATPase subunit B in the zebrafish Danio rerio.

In the present study we tested the hypothesis that two isoforms of the regulatory subunit B of vacuolar-type ATPase (V-ATPase) are expressed in the zebrafish Danio rerio. The complete coding sequences for both isoforms, vatB1 and vatB2, were cloned and sequenced. BLASTX analysis revealed the greatest similarity to amino acid sequences of B subunits from the European eel Anguilla anguilla and rainbow trout Oncorhynchus mykiss. The isoforms were expressed in a bacterial system and the recombinant proteins verified using isoform-specific antibodies directed against vatB isoforms of the eel. The distribution of both isoforms in zebrafish tissues was investigated using reverse transcriptase-polymerase chain reaction and western blot analysis. The results revealed that at the RNA level both isoforms were expressed in all tested organs, i.e. the gills, swimbladder, heart, kidney, liver, spleen, intestine and skeletal muscle. At the protein level, however, there were tissue-specific variations in the levels of the two vatB isoforms expressed. The highest amounts of V-ATPase were detected in total protein preparations from gill, heart and liver tissue. In liver tissue, however, the western blot analysis indicated that vatB1 was not as prominent as vatB2, and immunohistochemistry revealed that antibodies directed against vatB1 yielded a very weak staining in a number of cells, while an antibody directed against vatB1 and vatB2 yielded a strong staining in virtually every cell. Similarly, neurosecretory cells of the small intestine were stained with an antibody directed against vatB1 and vatB2, but not with an antibody specific for vatB1. Therefore we conclude that the differential expression of two isoforms of the V-ATPase subunits, which may serve different functions as in several mammalian species, may also be a common phenomenon in teleost fish.

Localization of the vacuolar-type ATPase in swimbladder gas gland cells of the European eel (Anguilla anguilla).

The vacuolar ATPase is a multifunctional enzyme that consists of several subunits. Subunit B is part of the catalytic domain of the enzyme and is present in two isoforms in fish as well as in mammals. Possibly, these two isoforms - vatB1 (kidney isoform) and vatB2 (brain isoform) - serve different functions. A localization of the two isoforms was attempted in swimbladder gas gland cells of the European eel Anguilla anguilla by immunohistochemistry. Two antibodies were produced by immunization of rabbits with synthetic peptides. Specificity of the antibodies, on the one hand, an isoform-specific antibody for vatB1 and, on the other hand, an antibody that recognizes both isoforms (vatB1 and vatB2), was confirmed by western blot analysis using recombinant proteins produced in a bacterial expression system. The immunohistochemical localization with the antibody directed against both isoforms of the B subunit revealed a positive staining in apical membranes of swimbladder gas gland cells as well as in the basolateral membranes. Significant staining was observed in vesicles located near the apical membrane. Staining with the vatB1-specific antibody resulted in a similar picture in the apical region of the cells. In contrast to the staining with the first antibody, only a poor signal was observed in the basal region. The nature of the vesicles in the apical region of the gas gland cells was determined by using an antibody directed against surfactant protein D.

Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae.

Cardiac activity and anaerobic metabolism were analyzed in zebrafish larvae raised under normoxia (PO(2) = 20 kPa) and under chronic hypoxia (PO(2) = 10 kPa) at three different temperatures (25, 28, and 31 degrees C). Heart rate increased with development and with temperature. Under normoxia, cardiac output increased significantly at high temperature (31 degrees C), but not at 28 or at 25 degrees C. Under chronic hypoxia, however, heart rate as well as cardiac output increased at all temperatures in larvae at about hatching time or shortly thereafter. Cardiac activity of larvae raised for 2 wk after fertilization with a reduced hemoglobin oxygen-carrying capacity in their blood (hypoxemia; due to the presence of CO or of phenylhydrazine in the incubation water) was not different from control animals. Whole body lactate content of these animals did not increase. Thus there was no indication of a stimulated anaerobic energy metabolism. The increase in cardiac activity observed during hypoxia suggests that at about hatching time receptors are present that sense hypoxic conditions, and this information can be used to induce a stimulation of convective oxygen transport to compensate for a reduction in bulk oxygen diffusion in the face of a reduced oxygen gradient between environmental water and tissues. Under normoxia, however, the PO(2) gradient between environmental water and tissues and diffusional oxygen transport assure sufficient oxygen supply even if hemoglobin oxygen transport in the blood is severely impaired. Thus, under normoxic conditions and with a normal metabolic rate of the tissues, convective oxygen transport is not required until approximately 2 wk after fertilization.

Mechanisms of acid secretion in pseudobranch cells of rainbow trout (Oncorhynchus mykiss).

Cell suspensions of rainbow trout Oncorhynchus mykiss pseudobranch, prepared by Ca(2+) depletion and mechanical maceration, contained a distinct population of cells that always kept their relatively cuboidal shape and did not round up in suspension or proliferate after adhering to the surface of cell culture dishes. Phasecontrast microscopy revealed an extensive system of basal membrane invaginations, and Na(+)-K(+)-ATPase- and anionexchanger-like immunoreactivity could be localized in cell membranes. The cells were characterized by a high mitochondrial density. Using specific antibodies, V-ATPase subunit B was localized in the plasma membrane. Using a cytosensor microphysiometer, the rate of acid secretion of these cells was measured and compared with the activity of a gill cell preparation. Incubation of pseudobranch cells with bafilomycin A1 (10(-6) mol l(-1)), a specific inhibitor of V-ATPase, reduced the rate of acid secretion by about 10% under control conditions, while no effect of bafilomycin on the rate of acid secretion of gill cells was observed. Application of amiloride (5 x 10(-5) mol l(-1)) reduced the rate of acid secretion in cells of both organs, pseudobranch and gills. Incubation of pseudobranch cells with DIDS (10(-3) mol l(-1)) resulted in a minor increase in the rate of proton secretion, but in cells prepared from the gills of rainbow trout acid secretion was reduced by about 30-40%. It is concluded that pseudobranch cells are equipped with various pathways to secrete protons, and that the anion exchange activity especially of pseudobranch cells appears to be different from that in gills.

Determinants of intracellular pH in gas gland cells of the swimbladder of the European eel Anguilla anguilla.

Gas gland cells of the European eel (Anguilla anguilla) were cultured on collagen-coated coverslips, and intracellular pH was measured using the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxypropyl)-5-(6)-carboxyfluorescein (BCPCF). The contributions of various proton-translocating mechanisms to homeostasis of intracellular pH (pHi) were assessed by adding specific inhibitors of the various proton-translocating mechanisms at a constant extracellular pH (pHe) of 7.4 and after artificial acidification of the cells using the ammonium pulse technique. The greatest decrease in pHi was observed after addition of 5-(N-ethyl-N-isobutyl)-amiloride (MIA), an inhibitor of Na(+)/H(+) exchange. Na(+)/H(+) exchange was active under steady-state conditions at an extracellular pH of 7.4, and activity increased after intracellular acidification. Incubation of gas gland cells with 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of anion exchange, also caused a decrease in pHi, but this decrease was not as pronounced as in the presence of MIA. Furthermore, at low pHi, the effect of DIDS was further reduced, suggesting that bicarbonate-exchanging mechanisms are involved in maintaining a steady-state pHi but that their importance is reduced at low pH. Bafilomycin A(1), a specific inhibitor of the V-ATPase, had no effect on steady-state pHi. However, recovery of intracellular pH after an artificial acid load was significantly impaired in the presence of bafilomycin. Our results suggest that Na(+)/H(+) exchange and anion exchange are important for the regulation of pHi at alkaline values of pHe. When pHi is low, a situation probably often encountered by gas gland cells during gas secretion, Na(+)/H(+) exchange continues to play an important role in acid secretion and a V-ATPase appears to contribute to proton secretion.

Swimbladder gas gland cells cultured on permeable supports regain their characteristic polarity.

A cell culture system has been developed in which swimbladder gas gland cells from the European eel (Anguilla anguilla) were cultured on a permeable support. Cells seeded on Anodisc 13 (Whatman) or Costar Transwell 13 mm membranes form a confluent cell layer within the first 2 or 3 days of culture but, on the basis of measurements of transepithelial resistance, it is a "leaky" cell layer. In a superfusion system, the apical and basal sides of the cells were superfused asymmetrically, with saline on the apical side and a glucose-containing cell culture medium on the basal side. Under these conditions, the cells continuously produced lactic acid, and approximately 60-70 % of this lactate was released at the basal side. To mimic the in vivo situation, the saline solution supplied to the apical side was replaced by humidified air in an additional series of experiments. Cells cultured in an air/liquid system produced even more lactate, and this lactate was only released to the basal side; there was no leakage of fluid to the apical side. After 4 or 5 days in the superfusion system, the cells were fixed for histological examination. The cells were columnar, similar to gas gland cells in vivo, and showed a clear polarity, with some small microvilli at the apical membrane and extensive membrane foldings at lateral and basal membranes. Immunohistochemical localization of Na+/K+ -ATPase revealed that this ATPase was present mainly in the lateral membranes; it was never found in the apical membranes. Cells cultured in the air/liquid system showed a similar structure and polarity.

Growth and metabolism of larval zebrafish: effects of swim training.

Larval zebrafish (Danio rerio) of three different age classes ('yolk-sac' larvae, 96 h; 'swim-up' larvae, 9 days old; and 'free-swimming' larvae, 21 days old) were trained for 2, 6 and 11 days, respectively, to swim at 0 body lengths per second (BL s(-1)), 2 BL s(-1) and 5 BL s(-1). Survival was significantly diminished in larvae trained at 5 BL s(-1) compared to controls (0 BL s(-1)). Although training produced no significant differences in mass and length, the youngest larvae absorbed their yolk at a faster rate during training. Routine oxygen consumption ((MO(2)r)) and mass-specific routine oxygen consumption ((MO(2)r,m)) were not significantly affected by chronic training in the yolk-sac larvae and swim-up larvae. However, trained free-swimming larvae had a significantly higher (MO(2)r) (after 11 days of training) and (MO(2)r,m) (after 8 and 11 days of training) compared to control larvae. Trained free-swimming larvae consumed significantly less oxygen during swimming compared to control larvae, as measured by closed-system respirometry. Trained yolk-sac larvae exposed to increasing hypoxia levels were more effective O(2) regulators. Additionally, training enhanced survival during exposure to extreme hypoxia in all age groups. Thus, physiological acclimation to chronic swimming occurs in the earliest stages of life in the zebrafish.

Infection of the glass-eel swimbladder with the nematode Anguillicola crassus.

The ability of the nematode Anguillicola crassus to infect eel larvae (glass-eel stage) was tested. The results show that glass-eels fed on infected copepods, the natural intermediate host of the nematode, can be infected. Light microscopical examination of the infected developing swimbladder tissue revealed that the infection results in a significant thickening of the connective tissue. The basolateral labyrinth of gas gland cells is very much reduced in infected swimbladders, and the distance of gas gland cells to blood capillaries is enlarged. Critical swimming speed, defined as the speed where the larvae were no longer able to swim against the current, was similar in infected and uninfected animals. At intermediate speeds (about 60-80% of critical swimming speed) infected eels showed a slightly higher swimming activity than control animals. Resting oxygen consumption, measured as an index of metabolic activity, within the first 2 months of infection was higher in control animals, which may be due to a reduced rate of activity in infected glass-eels. By 4-5 months after the infection, however, it was significantly higher in infected animals. This may indicate that at this stage a higher activity of the animals is required to compensate for the increase in body density, but swimming performance of infected and non-infected glass-eels was not significantly different. Oxygen consumption during swimming activity, measured in a swim tunnel at 50% of maximal swimming speed, also was not affected. The results thus show that even glass-eels can be infected with A. crassus, and this probably contributes to the rapid spread of the nematode in Europe. While aerobic metabolism during swimming activity is not affected at this stage of infection, the swimbladder tissue shows severe histological changes, which most likely will impair swimbladder function.

Nitric oxide and vascular reactivity in developing zebrafish, Danio rerio.

We used a newly developed digital motion analysis video technique to study the effects of nitric oxide (NO) and epinephrine on the early larval arterial and venous vasculature of zebrafish. Application of the NO donor sodium nitroprusside resulted in a significant increase in both the venous and arterial vessel diameters, whereas N(G)-nitro-L-arginine methyl ester caused a significant decrease in the same diameters. Thus our results show that both the venous and arterial vasculature of the 5- and 6-day-old zebrafish larvae are influenced by endogenously produced NO. By use of immunohistochemistry, NO synthase immunoreactivity was demonstrated in endothelial cells of the dorsal vein. Local application of epinephrine onto the dorsal artery had no effect on vessel diameter. However, if the embryos were preincubated with N(omega)-nitro-L-arginine methyl ester, addition of epinephrine resulted in a significant reduction in both arterial and venous vessel diameters. Thus this study provides increasing evidence that before a functional autonomic innervation of the peripheral vascular system, vascular tone in larval tissue is regulated by a complex interaction of vasoactive substances that are produced locally by vascular endothelial cells.

Swim bladder gas gland cells produce surfactant: in vivo and in culture.

Electron microscopical examination of gas gland cells of the physostome European eel (Anguilla anguilla) and of the physoclist perch (Perca fluviatilis) revealed the presence of significant numbers of lamellar bodies, which are known to be involved in surfactant secretion. In the perch, in which the gas gland is a compact structure and gas gland cells are connected to the swim bladder lumen via small canals, lamellar bodies were also found in flattened cells forming the swim bladder epithelium. Flat epithelial cells are absent in the eel swim bladder, in which the whole epithelium consists of cuboidal gas gland cells. In both species, Western blot analysis using specific antibodies to human surfactant protein A (SP-A) showed a cross-reaction with swim bladder tissue homogenate proteins of approximately 65 kDa and in the eel occasionally of approximately 120 kDa, probably representing SP-A-like proteins in a dimeric and a tetrameric state. An additional band was observed at approximately 45 kDa. Western blots using antibodies to rat SP-D again resulted in a single band at approximately 45 kDa in both species, suggesting that there might be a cross-reaction of the antibody to human SP-A with an SP-D-like protein of the swim bladder tissue. To localize the surfactant protein, eel gas gland cells were cultured on permeable supports. Under these conditions, the gas gland cells regain their characteristic polarity. Electron microscopy confirmed the presence of lamellar bodies in cultured cells, and occasionally, exocytotic events were observed. Immunohistochemical staining using an antibody to human SP-A demonstrated the presence of surfactant protein only in luminal membranes and in adjacent lateral membranes. Only occasionally, evidence was found for the presence of surfactant protein in lamellar bodies.

Temperature-dependent development of cardiac activity in unrestrained larvae of the minnow Phoxinus phoxinus.

The minnow (Phoxinus phoxinus) was raised up to the stage of swim bladder inflation at temperatures between 10 degrees C and 25 degrees C, and the time of development significantly decreased at higher temperatures. Accordingly, initiation of cardiac activity was observed at day 2 in 25 degrees C animals and at day 4 in 12.5 degrees C animals. Only a minor increase in body mass was observed during the incubation period, and, at the end of the incubation period, animals raised at 25 degrees C did not have a significantly lower body mass compared with animals raised at 15 degrees C. Metabolic activity, determined as the rate of oxygen consumption of a larva, increased from 3.3 to 19.5 nmol/h during development at 15 degrees C and from 5.6 to 47.6 nmol/h during development at 25 degrees C. Heart rate showed a clear correlation to developmental stage as well as to developmental temperature, but at the onset of cardiac activity, diastolic ventricular volume and also stroke volume were higher at the lower temperatures. Furthermore, stroke volume increased with development, except for the group incubated at 12.5 degrees C, in which stroke volume decreased with development. Initial cardiac output showed no correlation to incubation temperature. Although metabolic activity increased severalfold during development from egg to the stage of swim bladder inflation at 15 degrees C and at 25 degrees C, weight-specific cardiac output increased only by approximately 40% with proceeding development. At 12.5 degrees C, cardiac output remained almost constant until opening of the swim bladder. The data support the notion that oxygen transport is not the major function of the circulatory system at this stage of development. The changes in heart rate with temperature appear to be due to the intrinsic properties of the pacemaker; there was no indication for a regulated response.

Digital motion analysis as a tool for analysing the shape and performance of the circulatory system in transparent animals.

The analysis of perfusion parameters using the frame-to-frame technique and the observation of small blood vessels in transparent animals using video microscopy can be tedious and very difficult because of the poor contrast of the images. Injection of a fluorescent probe (fluorescein isothiocynate, FITC) bound to a high-molecular-mass dextran improved the visibility of blood vessels, but the gray-scale histogram showed blurring at the edges of the vessels. Furthermore, injection of the fluorescent probe into the ventricle of small zebrafish (Danio rerio) embryos (body mass approximately 1 mg) often resulted in reduced cardiac activity. Digital motion analysis, however, proved to be a very effective tool for analysing the shape and performance of the circulatory system in transparent animals and tissues. By subtracting the two fields of a video frame (the odd and the even frame), any movement that occurred within the 20 ms necessary for the acquisition of one field could be visualised. The length of the shifting vector generated by this subtraction, represented a direct measure of the velocity of a moving particle, i.e. an erythrocyte in the vascular system. By accumulating shifting vectors generated from several consecutive video frames, a complete trace of the routes over which erythrocytes moved could be obtained. Thus, a cast of the vascular system, except for those tiny vessels that are not entered by erythrocytes, could be obtained. Because the gray-scale value of any given pixel or any given group of pixels increased with the number of erythrocytes passing it, digital motion analysis could also be used to visualise the distribution of blood cells in transparent tissues. This method was used to describe the development of the peripheral vascular system in zebrafish larvae up to 8 days post-fertilisation. At this stage, food intake resulted in a clear redistribution of blood between muscle tissue and the gut, and alpha-adrenergic control of peripheral blood flow was established.

Expression of two vacuolar-type ATPase B subunit isoforms in swimbladder gas gland cells of the European eel: nucleotide sequences and deduced amino acid sequences.

The poly(A)(+) RNA of swimbladder gas gland cells of the European eel Anguilla anguilla was isolated and used for cDNA synthesis. Using a pair of degenerate PCR primers directed towards the evolutionary highly conserved central part of the B subunit of vacuolar type H(+)-ATPase (V-ATPase) a fragment of 388 bp was amplified. By sequencing the cloned PCR products two different amplicons with a sequence identity of about 86% were obtained. BLASTN searches revealed a high degree of similarity of both to V-ATPase B subunits of other species. The sequences were completed by performing rapid amplification of cDNA ends PCR, subsequent cloning, and sequencing of the obtained products. The expression of two different isoforms of the V-ATPase B subunit is already demonstrated for Homo sapiens and Bos taurus. This is the first report that attributes the same phenomenon to a non-mammalian species, A. anguilla. The first isoform found in eel (vatB2) shows the highest degree of amino acid sequence homology with the human brain isoform (98.2%), the second one (vatB1) with the B subunit sequence of rainbow trout (Oncorhynchus mykiss) gill and kidney (98, 6%). The alignment of the deduced amino acid sequences of vatB1 and vatB2 shows that the highest sequence variation between these two isoforms is found at the amino-terminus, where vatB1 is nine amino acids shorter than vatB2, while at the carboxy-terminus it is two amino acids longer than vatB2. This has also been reported for the human and bovine kidney isoforms when compared with the brain isoforms. Northern blot analysis using specific hybridization probes revealed the expression of two mRNA's with lengths of about 2.9 kb and 3.5 kb for vatB1 and vatB2, respectively. For mammals, it is well known that V-ATPases containing the kidney isoforms of the B subunit are responsible for the extrusion of protons across the plasma membranes of several cell types. The fact that eel vatB1 seems to share structural features with the kidney isoforms in mammals supports the hypothesis that in gas gland cells a V-ATPase contributes to the acidification of the blood in the swimbladder.

Swimbladder gas gland cells of the European eel cultured in a superfusion system.

Swimbladder gas gland cells are polar epithelial cells which release acidic metabolites through the membranes of an extensive basolateral labyrinth, and secret surfactant via exocytosis at apical membranes. We have developed a method to establish primary cell cultures of gas gland cells in order to establish a model system for physiological analysis of gas gland cell function in vitro. Isolated gas gland cells attach to collagen S coated surfaces. Cells cultured in collagen S coated petri dishes were flat and showed no histological polarity. Cells cultured on Anodisc membranes in a superfusion system, in which the apical and basal side of the cells was supplied with a saline solution and with glucose containing DMEM cell culture medium, respectively, showed a clear polarity similar to the in vivo situation. Measurement of lactate release at the apical side and at the basal side revealed that these cells were functionally polar and secreted at least 70% of the lactate at their basal membranes. Gas gland cells could also be cultured in an air/liquid system, in which the apical membrane was exposed to humidified air. Cells cultured under these conditions released lactate only on the basal side and histologically were similar to cells cultured in the superfusion system.

Localization of carbonic anhydrase in swimbladder of European eel (Anguilla anguilla) and perch (Perca fluviatilis).

The distribution of carbonic anhydrase in swimbladder tissue and especially in gas gland cells of the European eel (Anguilla anguilla) and the perch (Perca fluviatilis) was analysed using histochemical staining according to Hansson (1967), with modifications proposed by Riddersträle (1991). While in the European eel, gas gland cells are distributed as a single layered epithelium over the whole secretory part of the swimbladder, the gas gland of the perch consists of a compact, richly vascularized 'multilayered' epithelium, in which gas gland cells have contact with the swimbladder lumen via small channels. In spite of these differences in organization, membranes of gas gland cells near blood vessels are richly folded in both species. A strong histochemical staining for carbonic anhydrase was observed in these membrane foldings. With prolonged incubation times a positive reaction was also observed in the cytoplasm of gas gland cells. In addition, the vascular endothelium and the erythrocytes showed a positive histochemical reaction. No staining, however, was visible in apical membranes towards the lumen of the swimbladder. In the perch, swimbladder epithelial cells outside the gas gland showed no positive staining of carbonic anhydrase. The results thus indicate that carbonic anhydrase activity is especially concentrated in membranes facing blood vessels. This suggests that a rapid equilibrium of the CO2/HCO3- reaction in the intracellular as well as in the extracellular space is essential for swimbladder function.

Vasodilation of swimbladder vessels in the european eel (Anguilla anguilla) induced by vasoactive intestinal polypeptide, nitric oxide, adenosine and protons

The effects of &bgr;-adrenergic stimulation, vasoactive intestinal polypeptide (VIP), adenosine, the nitric oxide (NO)-releasing agent sodium nitroprusside and of metabolic end-products of gas gland cell metabolism on swimbladder blood flow were investigated using saline- or blood-perfused swimbladder preparations of the freshwater European eel Anguilla anguilla. While &bgr;-adrenergic vasodilation was not detectable, a bolus injection of adenosine (100 microl, 10(-)7 mol l-1) and application of VIP (10(-)7 mol kg-1) caused a significant decrease in perfusion pressure in saline-perfused swimbladder preparations. Immunohistochemical analysis revealed the presence of VIP-immunoreactive nerve fibres in the swimbladder artery and in the swimbladder vein (seawater-adapted eels were used for immunohistochemical studies). Application of sodium nitroprusside also elicited a small, but significant, decrease in perfusion pressure in saline-perfused swimbladder preparations, while preincubation of swimbladder tissue with N(&ohgr;)nitro-l-arginine, a non-selective inhibitor of nitric oxide synthase, significantly enhanced the flow-induced increase in perfusion pressure. Lactate, the major metabolic end-product of gas gland cell metabolism, had no effect on perfusion pressure. In contrast, an increase in proton concentration in both saline- and blood-perfused preparations induced a vasodilation, as indicated by a significant decrease in perfusion pressure. The results demonstrate that VIP, NO, adenosine and protons may induce a vasodilation of swimbladder blood vessels. None of these effects, however, compares in time span with the previously described immediate, short-lasting vasodilation of swimbladder vessels elicited by pulse stimulation of the vagus nerve.

Environmental influences on the development of the cardiac system in fish and amphibians.

In poikilothermic animals body temperature varies with environmental temperature, and this results in a change in metabolic activity (Q10 of enzymatic reactions typically is around 2-3). Temperature changes also modify gas transport in body fluids. While the diffusion coefficient increases with increasing temperatures, physical solubility and also hemoglobin oxygen affinity decrease. Therefore, an increase in temperature typically requires adjustments in cardiac activity because ventilatory and convectional transport of respiratory gases usually are tightly coupled in adults in order to meet the oxygen demand of body tissues. Hypoxic conditions also provoke adaptations in the central circulatory system, like the hypoxic bradycardia, which has been described for many adult lower vertebrates, combined with an increase in stroke volume and peripheral resistance. In embryos and larvae the situation is much more complicated, because nervous control of the heart is established only late during development, and because the site of gas exchange changes from mainly cutaneous gas exchange during early development to mainly pulmonary or branchial gas exchange in late stages. In addition, recent studies in amphibian and fish embryos and larvae reveal, that at least in very early stages convectional gas transport of the hemoglobin is not essential, which means that in these early stages ventilatory and convectional gas transport are not yet coupled. Accordingly, in early stages of fish and amphibians the central cardiac system often does not respond to hypoxia, although in some species behavioral adaptations indicate that oxygen sensors are functional. If a depression of cardiac activity is observed, it most likely is a direct effect of oxygen deficiency on the cardiac myocytes. Regulated cardiovascular responses to hypoxia appear only in late stages and are similar to those found in adult species.