Metabolic fate of exogenous 15NH4Cl in the gulf toadfish (Opsanus beta)

This study was undertaken to determine whether gulf toadfish (Opsanus beta) could metabolize ammonia from their environment into other, less toxic products. To this end, gulf toadfish were exposed to 3.8 mM 15NH(4)Cl in seawater for 24 and 48 h. Liver, kidney, gill, brain and muscle samples were analyzed for distribution of 15N within the tissue and among various nitrogen-containing metabolites (ammonia, amino-N, glutamine-N, urea and protein). The data reported here show that the toadfish can indeed take up and metabolize ammonia. Analysis of individual metabolic products of ammonia indicates that the toadfish can convert this toxic chemical into other less toxic metabolites. Ammonia enrichment is significantly different over controls in the kidney, brain and muscle. Urea enrichment is most significant in the brain, with less significant enrichment occurring in the liver and muscle. While accumulation of ammonia into an amino acid pool was not a significant metabolic fate, protein synthesis was significantly enriched in all tissues (with the highest levels occurring in the gill) indicating that amino acid synthesis may be a pathway of ammonia detoxification en route to protein synthesis, and that environmental ammonia can be 'fixed' into protein. Finally, it was found that glutamine-N synthesis occurs at significant levels in the liver, brain and muscle.     

Ocean acidification and the loss of phenolic substances in marine plants

Rising atmospheric CO(2) often triggers the production of plant phenolics, including many that serve as herbivore deterrents, digestion reducers, antimicrobials, or ultraviolet sunscreens. Such responses are predicted by popular models of plant defense, especially resource availability models which link carbon availability to phenolic biosynthesis. CO(2) availability is also increasing in the oceans, where anthropogenic emissions cause ocean acidification, decreasing seawater pH and shifting the carbonate system towards further CO(2) enrichment. Such conditions tend to increase seagrass productivity but may also increase rates of grazing on these marine plants. Here we show that high CO(2) / low pH conditions of OA decrease, rather than increase, concentrations of phenolic protective substances in seagrasses and eurysaline marine plants. We observed a loss of simple and polymeric phenolics in the seagrass Cymodocea nodosa near a volcanic CO(2) vent on the Island of Vulcano, Italy, where pH values decreased from 8.1 to 7.3 and pCO(2) concentrations increased ten-fold. We observed similar responses in two estuarine species, Ruppia maritima and Potamogeton perfoliatus, in in situ Free-Ocean-Carbon-Enrichment experiments conducted in tributaries of the Chesapeake Bay, USA. These responses are strikingly different than those exhibited by terrestrial plants. The loss of phenolic substances may explain the higher-than-usual rates of grazing observed near undersea CO(2) vents and suggests that ocean acidification may alter coastal carbon fluxes by affecting rates of decomposition, grazing, and disease. Our observations temper recent predictions that seagrasses would necessarily be "winners" in a high CO(2) world.     

HCO3-stimulated Cl efflux in the gulf toadfish acclimated to sea water.

Unidirectional efflux of Cl was examined in the Gulf toadfish, Opsanus beta, in artificial seawater solutions with modified concentrations of Cl and HCO3. Removal of Cl HCO3 reduced Cl efflux. Addition of HCO3 at typical seawater concentrations stimulated Cl efflux, independent of changes in the transepithelial potential. This active, HCO3-stimulated Cl efflux is saturable, with a Km of 2.4 mM, typical of the concentration of HCO3 found in sea water, and independent of external pH. Active extrusion of Cl offsets the net diffusional and oral gain of Cl faced by O. beta in sea water.     

5-Hydroxytryptamine initiates pulsatile urea excretion from perfused gills of the gulf toadfish (Opsanus beta)

When stressed, toadfish become ureotelic and excrete almost all of their nitrogenous waste in 1-3 daily pulses of urea-N across the gills. Intravascular injections of 5-hydroxytyptamine (5-HT; serotonin) and analogues also elicit marked excretory pulses of urea-N from toadfish in vivo, suggesting that 5-HT release is the proximate trigger for spontaneous pulses. However it is unclear whether 5-HT is acting on the gills directly or elsewhere to cause the effect indirectly. A perfused whole gill preparation which maintained normal pressure relationships and stable vascular resistance was employed to address this question. Bolus injections into the ventral aortic perfusate of either 5-HT (1, 10 μmol kg(-1)) or the specific 5-HT(2) receptor agonist α-methyl 5-HT (1, 10 μmol kg(-1)) elicited rapid urea-N pulses from perfused toadfish gills. The effective doses, the post-injection delays (5.5 ± 1.3 min, range=2-22), the percent occurrences (57-85%), and the magnitude of the induced urea-N pulses (615.4 ± 131.3 μmol-N kg(-1), range 66.0-2634.0), were all similar to those previously reported when these agents were injected in vivo. Bolus injections of 5-HT and α-methyl 5-HT also elicited a biphasic response in ventral aortic pressure, reflecting an initial rapid short-lived vasodilation and a subsequent longer-lasting vasoconstriction. These events were similar to those which have been recorded to occur at a greater frequency during spontaneous urea-N pulsing in vivo. Neither the urea-N pulsing nor the cardiovascular responses to 5-HT were inhibited by the 5-HT(2A) receptor subtype blocker, ketanserin (pre-injection with 10 μmol kg(-1) plus 33 μmol L(-1) in the perfusate). Overall, these results provide strong support for the idea that the proximate stimulus for natural urea pulsing in vivo is 5-HT mobilization, acting directly in the gills.     

Pulsatile urea excretion in the gulf toadfish: mechanisms and controls

Opsanus beta expresses a full complement of ornithine–urea cycle (OUC) enzymes and is facultatively ureotelic, reducing ammonia-N excretion and maintaining urea-N excretion under conditions of crowding/confinement. The switch to ureotelism is keyed by a modest rise in cortisol associated with a substantial increase in cytosolic glutamine synthetase for trapping of ammonia-N and an upregulation of the capacity of the mitochondrial OUC to use glutamine-N. The entire day's urea-N production is excreted in 1 or 2 short-lasting pulses, which occur exclusively through the gills. The pulse event is not triggered by an internal urea-N threshold, is not due to pulsatile urea-N production, but reflects pulsatile activation of a specific branchial excretion mechanism that rapidly clears urea-N from the body fluids. A bidirectional facilitated diffusion transporter, with pharmacological similarity to the UT-A type transporters of the mammalian kidney, is activated in the gills, associated with an increased trafficking of dense-cored vesicles in the pavement cells. An 1814 kB cDNA (‘tUT’) coding for a 475–amino acid protein with approximately 62% homology to mammalian UT-A's has been cloned and facilitates phloretin-sensitive urea transport when expressed in Xenopus oocytes. tUT occurs only in gill tissue, but tUT mRNA levels do not change over the pulse cycle, suggesting that tUT regulation occurs at a level beyond mRNA. Circulating cortisol levels consistently decline prior to a pulse event and rise thereafter. When cortisol is experimentally clamped at high levels, natural pulse events are suppressed in size but not in frequency, an effect mediated through glucocorticoid receptors. The cortisol decline appears to be permissive, rather than the actual trigger of the pulse event. Fluctuations in circulating AVT levels do not correlate with pulses; and injections of AVT (at supraphysiological levels) elicit only minute urea-N pulses. However, circulating 5-hydroxytryptamine (5-HT) levels fluctuate considerably and physiological doses of 5-HT cause large urea-N pulse events. When the efferent cranial nerves to the gills are sectioned, natural urea pulse events persist, suggesting that direct motor output from the CNS to the gill is not the proximate control.     

Intestinal zinc uptake in two marine teleosts,squirrelfish (Holocentrus adscensionis) and gulf toadfish (Opsanus beta)

Zinc is a vital micronutrient, yet as an environmental toxicant it can be deleterious to aquatic organisms such as fish. Consequently, the study of zinc uptake mechanisms is essential for understanding nutrition, toxicity, and metabolism of this metal. Intestinal zinc uptake was studied in two marine teleosts, using both in vitro (in vitro perfusion and intestinal sacs) and in vivo techniques (in situ bolus). Female squirrelfish (Holocentrus adscensionis) exhibited significantly increased epithelial zinc uptake associated with enhanced hepatic zinc accumulation. This confirms this zinc-hyperaccumulating teleost as a potential model of zinc absorption. Intestinal zinc uptake in the gulf toadfish (Opsanus beta) was biphasic with respect to zinc concentration (0.3-500 microM), exhibiting both saturable and passive uptake components. In both species, the passage of zinc into the postintestinal compartment was highly dependent on technique. Decreased proportions of postintestinal zinc in vivo, coupled with concentration-dependent distribution of zinc accumulation, suggested mechanisms may act to control the movement of zinc into the circulation. In addition, the results of this study were used to reinterpret previous findings of zinc uptake in freshwater fish and allowed a critique of techniques used to study intestinal metal uptake.     

Structure and function of the axillary organ of the gulf toadfish, Opsanus beta (Goode and Bean)

The structure of the axillary organ of a batrachoidid species, the gulf toadfish (Opsanus beta Goode and Bean 1879), has been examined and several simple experiments designed to elucidate its function performed. Electron microscopy (EM) studies revealed cells and structures suggesting secretory and iono regulatory roles (e.g., abundant intracytoplasmic secretory particles, rough endoplasmic reticulum, sparse Golgi bodies, indented epithelial cells with microvilli, numerous endocytotic vesicles, etc.). Our physiological experiments allowed us to reach several conclusions: the organs do not excrete significant quantities of urea relative to other areas of the fish (head and gills), the organs do not secrete a substance that is toxic to a teleost test fish (Gambusia affinis), the secretions do not induce short-term modifications in locomotory activity of other gulf toadfish (e.g., by pheromonal means) and the secretions do not inhibit the growth of several species of microorganisms in culture. The function of the organ and its secretions remains unknown, representing a fertile area for research on structure and function in comparative physiology.     

Ocean acidification accelerates net calcium carbonate loss in a coral rubble community

Coral rubble communities are an important yet often overlooked component of a healthy reef ecosystem. The organisms inhabiting reef rubble are primarily bioeroders that contribute to the breakdown and dissolution of carbonate material. While the effects of ocean acidification on calcifying communities have been well studied, there are few studies investigating the response of bioeroding communities to future changes in pH and calcium carbonate saturation state. Using a flow-through pH-stat system, coral rubble pieces with a naturally occurring suite of organisms, along with bleached control rubble pieces, were subjected to three different levels of acidification over an 8-week period. Rates of net carbonate loss in bleached control rubble doubled in the acidification treatments (0.02 vs. 0.04% CaCO₃ d^?1 in ambient vs. moderate and high acidification), and living rubble communities experienced significantly increased rates of net carbonate loss from ambient to high acidification conditions (0.06 vs. 0.10% CaCO₃ d^?1, respectively). Although more experimentation is necessary to understand the long-term response and succession of coral rubble communities under projected conditions, these results suggest that rates of carbonate loss will increase in coral rubble as pH and calcium carbonate saturation states are reduced. This study demonstrates a need to thoroughly investigate the contribution of coral rubble to the overall carbonate budget, reef resilience, recovery, and function under future conditions.     

Comparison of the effects of ammonia on brain mitochondrial function in rats and gulf toadfish

We compared the effect of hyperammonemia on NADH levels in brain slices and on the rate of oxygen consumption from isolated nonsynaptic brain mitochondria in ammonia-sensitive Wistar rats with that in ammonia-tolerant gulf toadfish (Opsanus beta). The NADH content was significantly decreased (12% less than control after 45 min with 1 mM NH(4)Cl) in rat brain slices, but it was not affected in brain slices from toadfish (with both 1 and 6 mM NH(4)Cl). The rates of oxygen consumption of different sets of enzymes of the electron transport chain (ETC; complexes I, II, III, and IV; II, III, and IV; and IV alone) were unaltered by hyperammonemic conditions in isolated nonsynaptic mitochondria from either rats or toadfish. These results lead us to conclude that the differing effects of ammonia on NADH levels in rat and toadfish brain slices must be due to aspects other than the direct effects of ammonia on enzymes of the ETC. Additionally, because these effects were seen in vitro, our studies enabled us to rule out the possibility that effects of ammonia on metabolism were via indirect systemic effects. These results are discussed in the context of current views on mechanisms of central nervous system damage in hyperammonemic states.     

Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta

Summary The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however porcine brain natriuretic peptidelike immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum. Many immunoreactive fibres were present in the rostral regions of the inferior lobes of the hypothalamus and in the dorsolateral and ventrolateral aspects of the rhombencephalon. No immunoreactivity was observed in the heart and brain using rat atrial natriuretic and eel natriuretic peptide antisera. Although the chemical structure of natriuretic peptides in the heart and brain of toadfish is unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic and/or porcine C-type natriuretic peptides. The presence of natriuretic peptides in the brain suggests that they could be important neuromodulators and/or neurotransmitters.     

Immunohistochemical localization of urea and ammonia transporters in two confamilial fish species, the ureotelic gulf toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus)

This study aims to illustrate potential transport mechanisms behind the divergent approaches to nitrogen excretion seen in the ureotelic toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus). Specifically, we wish to confirm the expression of a urea transporter (UT), which is found in the gill of the toadfish and which is responsible for the unique “pulsing” nature of urea excretion and to localize the transporter within specific gill cells and at specific cellular locations. Additionally, the localization of ammonia transporters (Rhesus glycoproteins; Rhs) within the gill of both the toadfish and midshipman was explored. Toadfish UT (tUT) was found within Na⁺-K⁺-ATPase (NKA)-enriched cells, i.e., ionocytes (probably mitochondria-rich cells), especially along the basolateral membrane and potentially on the apical membrane. In contrast, midshipman UT (pnUT) immunoreactivity did not colocalize with NKA immunoreactivity and was not found along the filaments but instead within the lamellae. The cellular location of Rh proteins was also dissimilar between the two fish species. In toadfish gills, the Rh isoform Rhcg1 was expressed in both NKA-reactive cells and non-reactive cells, whereas Rhbg and Rhcg2 were only expressed in the latter. In contrast, Rhbg, Rhcg1 and Rhcg2 were expressed in both NKA-reactive and non-reactive cells of midshipman gills. In an additional transport epithelium, namely the intestine, the expression of both UTs and Rhs was similar between the two species, with only subtle differences being observed.     

Diel patterns of nitrogen excretion, plasma constituents, and behavior in the gulf toadfish (Opsanus beta) in laboratory versus outdoor mesocosm settings

Nitrogen excretion by the gulf toadfish (Opsanus beta) is of interest because of its high proportion of urea excretion compared with that of other teleosts. To better understand the factors influencing the timing of nitrogen excretion, the ratio of excreted urea∶ammonia, and the effector molecules regulating these processes, gulf toadfish were subjected to a series of experiments that moved them progressively from internal laboratory to outdoor mesocosm settings while assessing their behavior, nitrogen excretion patterns, levels of plasma hormones/effectors, and other parameters. In confined flux chambers in both laboratory and outdoor settings, toadfish nitrogen excretion was largely observed as urea pulses, with no apparent diel patterns to the pulses. Unrestrained toadfish in mesocosms exhibited distinctly nocturnal behavior, remaining exclusively in shelters during the day but taking several forays out into the mesocosm at night. In contrast to nitrogen excretion patterns in chambers, urea and ammonia were coexcreted in mesocosms and ratios for urea∶ammonia were very close to 1∶1 for both fed and fasted toadfish. The majority of measured excretion (and corresponding declines in plasma urea levels) occurred during two distinct periods of pulsing during daylight hours (0600-1000 and 1600-1800 hours). The declines in plasma urea associated with excretion were preceded by/coincided with declines in plasma cortisol. No day/night or hourly patterns in plasma serotonin (5-hydroxytryptamine [5-HT]) were observed, but there was a strong positive correlation among all samples between plasma urea and 5-HT. There was also a negative correlation between plasma cortisol and 5-HT. As expected for a nocturnally active species, plasma melatonin was significantly lower in daylight hours. A variety of enzyme activities (glutamine synthetase, glutaminase) and mRNA levels (glutamine synthetase, urea transporter, and Rhesus proteins) showed no significant variation over a diel cycle. Unlike prior laboratory studies, our results show that gulf toadfish in a natural setting have a distinctly diurnal pattern of nitrogen excretion and that ammonia and urea are coexcreted. The decline in plasma cortisol associated with urea pulses noted in prior laboratory studies was not as evident in the natural setting.     

Do circulating plasma AVT and/or cortisol levels control pulsatile urea excretion in the gulf toadfish (Opsanus beta)?

Previous work has shown that pulsatile urea excretion at the gills of the gulf toadfish is due to periodic activation of a facilitated diffusion transport system with molecular and pharmacological similarity to the UT-A transport system of the mammalian kidney. In mammals, AVP and glucocorticoids are two important endocrine regulators of this system. The present study focused on the potential role of circulating AVT (the teleost homologue of AVP) and cortisol levels as possible triggers for urea pulses. Long-term (34-84 h) monitoring of plasma levels by repetitive sampling at 2-h intervals from chronic cannulae in individual toadfish demonstrated that circulating AVT concentrations are low (10(-12)-10(-11) M), and show no relationship to the occurrence of natural urea pulses. In contrast, plasma cortisol levels decline greatly prior to natural pulses and rise rapidly thereafter. AVT injections into the caudal artery or ventral aorta elicited pulse events, but these were extremely small (1-10%) relative to natural pulses, and occurred only at unphysiological dose levels (10(-9) M in the plasma). AVP was a partial agonist, but isotocin, insulin-like growth factor-1, and atrial natriuretic peptide were without effect at the same concentration. Artificially raising plasma cortisol levels by cortisol injection tended to reduce responsiveness to AVT. Pharmacological reduction of plasma cortisol levels by metyrapone injection elicited small pulses similar to those caused by AVT. Following such pulse events, AVT was ineffective in inducing pulses. We conclude that decreases in circulating cortisol play an important permissive role in urea pulsing, but that circulating AVT levels are not involved.     

Ocean acidification with (de)eutrophication will alter future phytoplankton growth and succession

Human activity causes ocean acidification (OA) though the dissolution of anthropogenically generated CO₂ into seawater, and eutrophication through the addition of inorganic nutrients. Eutrophication increases the phytoplankton biomass that can be supported during a bloom, and the resultant uptake of dissolved inorganic carbon during photosynthesis increases water-column pH (bloom-induced basification). This increased pH can adversely affect plankton growth. With OA, basification commences at a lower pH. Using experimental analyses of the growth of three contrasting phytoplankton under different pH scenarios, coupled with mathematical models describing growth and death as functions of pH and nutrient status, we show how different conditions of pH modify the scope for competitive interactions between phytoplankton species. We then use the models previously configured against experimental data to explore how the commencement of bloom-induced basification at lower pH with OA, and operating against a background of changing patterns in nutrient loads, may modify phytoplankton growth and competition. We conclude that OA and changed nutrient supply into shelf seas with eutrophication or de-eutrophication (the latter owing to pollution control) has clear scope to alter phytoplankton succession, thus affecting future trophic dynamics and impacting both biogeochemical cycling and fisheries.     

Loss of guanylyl cyclase C (GCC) signaling leads to dysfunctional intestinal barrier

Background

Guanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.

Methodology/Principal Findings

Paracellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC−/−) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN−/−) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC−/− and UGN−/− mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC−/− mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC−/− and UGN−/− mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.

Conclusions/Significance

GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.     

Field studies on the ureogenic gulf toadfish, in a subtropical bay. II. Nitrogen excretion physiology

In order to examine the in situ nitrogen excretion physiology of gulf toadfish ( Opsanus beta ) (Fam. Batrachoididae), several biochemical and physiological measurements relating to urea synthesis and excretion were measured in samples taken from freshly collected gulf toadfish from a subtidal population in Biscayne Bay, Florida, U.S.A. This indirect appoach was used, instead of direct measurements of nitrogen excretion, because nitrogen excretion patterns of gulf toadfish are altered markedly during the first 24 h of capture disturbance or laboratory confinement. The values obtained for plasma cortisol levels, and the activities of hepatic ornithine-urea cycle enzymes, including glutamine synthetase (and its partitioning between cytosolic and mitochondrial compartments), suggest that gulf toadfish in Biscayne Bay may excrete a substantial portion of their waste nitrogen as urea. Also conducted were correlation analyses of several biotic variables (plasma [cortisol], enzyme activities, plasma [urea], hepatosomatic index, and plasma [Ca⁺⁺]) with several abiotic variables (temperature, salinity, depth and dissolved oxygen), and with collection site and season. Results of these analyses are discussed in the context of hypotheses to explain ureotely in this teleost fish.     

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish (Opsanus beta)

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.     

Extracellular acidification leads to reactive oxygene species formation in rat brain synaptosomes

Formation of reactive oxygen species in rat brain synaptosomes was studied using DCFDA fluorescent dye at lowered extracellular pH. It has been shown that decrease in pH value from 7.4 to 7.0 and up to 6.0 leads to increase of fluorescence that is indicative of oxidative stress. The effect is observed regardless of whether Ca ions are present in incubation medium or no. Acidification of the incubation medium induces quenching of fluorescence of previously oxidized form of the dye in experiments without synaptosomes This evidences that increase of dye fluorescence is really associated with reactive oxygen species accumulation. Thus, it has been demonstrated that pH declined up to 7.0 in the incubation medium is sufficient to induce the formation of reactive oxygen species in synaptosomes.