The mechanisms and costs of physiological and toxicological acclimation to waterborne silver in juvenile rainbow trout (Oncorhynchus mykiss)

Juvenile rainbow trout were exposed to 0, 0.1, 1, 3, and 5 micro g/l silver (Ag, as AgNO3) for 23 days. Specific growth rate, cumulative food consumption, food-conversion efficiency, and critical swimming speed (U(crit)) were significantly reduced during 5 micro g/l Ag exposure, demonstrating a physiological cost of silver acclimation. Only the 5 microg/l Ag treatment had significant cumulative mortality (5.2%). Fish were most susceptible to silver on days 5 and 15. Exposure to 5 microg/l Ag significantly lowered plasma Na+ and Cl- on days 5 and 10, but plasma ion concentration recovered thereafter. Unidirectional Na+ uptake and gill Na/K-ATPase activity were significantly inhibited by 3 and 5 microg/l Ag exposure. Na+ uptake was inhibited by 3 micro g/l Ag at day 5 alone, whereas the effects at the highest Ag exposure persisted until day 15. Gill Na/K-ATPase was inhibited on day 5 in both the 3 and 5 microg/l Ag treatments but increased to approx. 1.5 times of control levels by day 23. Only the 3 and 5 microg/l Ag treatments produced toxicological acclimation (at least twofold elevations in 168-h LC50 values in fish subsampled on days 15 and 23). We conclude that physiological acclimation results from compensatory changes in Na+ transport at the gills, and that these changes may eventually lead to toxicological acclimation.     

Intestinal transport following transfer to increased salinity in an anadromous fish (Oncorhynchus mykiss)

The ability to transition from freshwater to seawater environments is an intrinsic requirement of the life history of some fish species, including the anadromous rainbow trout (Oncorhynchus mykiss). The differences between hyper- and hypoosmoregulation are developed quickly (in hours to days), and at all scales, from gene expression to organ function. In this study, intestinal ion and water transport was examined in O. mykiss following acute transfer from freshwater (FW) to 70% seawater (SW). Plasma [Mg2+] increased at 24h post-transfer but recovered by 72 h. In the intestinal fluids, total CO? was found to increase with SW exposure/acclimation, while [Na+] decreased after 24h of SW exposure. Overall, in vitro experiments demonstrated the importance of base secretion to epithelial water uptake, and suggested that the primary physiological adjustments occurred 24-72 h after acute SW transfer. The mRNA expression of ion transporters important for intestinal osmoregulation and maintenance of acid-base balance was also investigated. A Na+/H+ exchanger (NHE2) and anion exchanger (SLC26a6) were hypothesized to be involved in the transport of acid-base equivalents, Na+, and Cl?, but were not uniformly expressed across tissue samples, and expression, where present, did not change following salinity transfer. NHE1, however, was expressed in all examined tissues (gill, kidney, anterior intestine, and pyloric cecae), but exhibited no changes in expression following acute salinity transfer.     

Time course analysis of the mechanism by which silver inhibits active Na+ and Cl- uptake in gills of rainbow trout

A time course analysis using (110m)Ag, (24)Na(+), and (36)Cl(-) examined gill silver accumulation and the mechanism by which waterborne silver (4.0 x 10(-8) M; 4.3 microg/l) inhibits Na(+) and Cl(-) uptake in gills of freshwater rainbow trout. Analyses of gill and body fluxes allowed calculation of apical uptake and basolateral export rates for silver, Na(+), and Cl(-). To avoid changes in silver bioavailability, flow-through conditions were used to limit the buildup of organic matter in the exposure water. For both Na(+) and Cl(-) uptake, apical entry, rather than basolateral export, was the rate-limiting step; Na(+) and Cl(-) uptake declined simultaneously and equally initially, with both uptakes reduced by approximately 500 nmol.g(-1).h(-1) over the 1st h of silver exposure. There was a further progressive decline in Na(+) uptake until 24 h. Carbonic anhydrase activity was inhibited by 1 h, whereas Na(+)-K(+)-ATPase activity was not significantly inhibited until 24 h of exposure. These results indicate that carbonic anhydrase inhibition can explain the early decline in Na(+) and Cl(-) uptake, whereas the later decline is probably related to Na(+)-K(+)-ATPase blockade. Contrary to previous reports, gill silver accumulation increased steadily to a plateau. Despite the rapid inhibition of apical Na(+) and Cl(-) uptake, apical silver uptake (and basolateral export) increased until 10 h, before decreasing thereafter. Thus silver did not inhibit its own apical uptake in the short term. These results suggest that reduced silver bioavailability is the mechanism behind the pattern of peak and decline in gill silver accumulation previously reported for static exposures to silver.     

Ionoregulatory development and the effect of chronic silver exposure on growth, survival, and sublethal indicators of toxicity in early life stages of rainbow trout (Oncorhynchus mykiss)

Rainbow trout embryos and larvae were continuously exposed, in a flow-through system, to 0, 0.1 microg/l (measured=0.098 +/- 0.002 microg/l) or 1.0 microg/l (measured=0.853+/-0.022 microg/l) total silver (as AgNO3) in moderately hard water (120 mg CaCO3/l, 0.70 mM Cl, 1.3 mg/l dissolved organic matter and 13.7 +/- 0.1 degrees C) from fertilization to I week post-hatch. The objectives of the study were to investigate the effects of chronic silver exposure on mortality, time to hatch and growth, and on sublethal physiological indicators of toxicity. Exposure to 1.0 microg/l total silver resulted in a small, but statistically significant, increase in mortality (16%) relative to controls (12%) but interestingly, resulted in an increased rate of growth (as indicated by larval weight, length and extractable protein) and ionoregulatory development over the duration of this study. Whole body unidirectional Na uptake (J(in)Na+) increased with silver exposure concentration (both 0.1 microg/l and 1.0 microg/l total silver) just prior to and following hatch, with up to a three-fold elevation in J(in)Na+ in the 1.0 microg/l treatment relative to controls. Qualitatively similar changes in whole body Na+,K-ATPase activity (per mg protein or per whole embryo or larvae) also occurred over this period. By 1 week post-hatch, there were no differences in J(in)Na among treatments and Na+,K+-ATPase activity levels in silver exposed groups were significantly reduced relative to controls. Within 2 days following hatch, there was an elevation in whole larval ammonia levels, while cortisol levels were elevated at 1 week post-hatch in the 1.0 microg/l treatment relative to controls. Ionoregulatory disturbance and elevations in both cortisol and ammonia have also been observed during acute silver exposure in adult rainbow trout, indicating that chronic and acute mechanisms of toxicity may be similar.     

Bicarbonate/chloride antiport in Vero cells: II. Mechanisms for bicarbonate-dependent regulation of intracellular pH

The rates of bicarbonate-dependent uptake and efflux of 22Na+ in Vero cells were studied and compared with the uptake and efflux of 36Cl-. Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten-fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na+ was much less affected by changes in pH. The bicarbonate-linked uptake of 22Na+ was dependent on internal Cl- but not on internal Na+. At a constant external concentration of HCO3-, the amount of 22Na+ associated with the cells increased when the internal concentration of HCO3- decreased and vice versa, which is compatible with the possibility that the ion pair NaCO3- is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36Cl- both in the absence and presence of Na+. At alkaline internal pH, HCO3- stimulated the efflux of 36Cl- from preloaded cells, while at acidic internal pH both Na+ and HCO3- were required to induce 36Cl- efflux. We propose a model for how bicarbonate-dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH(-)-equivalents in opposite directions across the plasma membrane.     

The effects of acid exposure on the ion regulation and seawater adaptation of coho salmon (Oncorhynchus kisutch) parrs and smolts

Smolts exhibited decreases in plasma Na+ levels after 7 days and lower Na+, K+-ATPase activities 14 days after acid exposure. Parrs exhibited decreased plasma Na+ after 24 hr acid exposure. Plasma Na+ increased and Na+, K+-ATPase decreased in smolts after transfer to seawater. Parrs exhibited increased plasma Na+ as well as Na+, K+-ATPase activity immediately after transfer to seawater. It was concluded that acid exposure prior to entry into seawater was detrimental to coho salmon with regard to the length of acid exposure and stage of development. A possible mechanism by which fish die from acid stress is inhibition of gill Na+, K+-ATPase concomitant with decreases in plasma Na+ levels.     

Effects of Monovalent Ions on the Transport of Noradrenaline Across the Plasma Membrane of Neuronal Cells (PC-12 Cells)

The dependence on Na+, K+, and Cl- of uptake and accumulation of [3H]noradrenaline was studied in plasma membrane vesicles isolated from PC-12 pheochromocytoma cells. Plasma membrane vesicles accumulated [3H]noradrenaline when an inward-directed gradient for Na+ and an outward-directed gradient for K+ were imposed across the vesicle membrane. Under these conditions, initial rates of uptake of [3H]noradrenaline were saturable (Km = 0.14 microM) and inhibited by a series of substrates and inhibitors of "uptake". The IC50 values were positively correlated with those for inhibition of uptake into intact PC-12 cells. Uptake and accumulation of [3H]noradrenaline in plasma membrane vesicles were absolutely dependent on external Na+ and Cl-; they were dependent on an inwardly directed gradient for Na+ but less dependent on an inwardly directed gradient for Cl-. Internal K+ strongly enhanced uptake and accumulation of [3H]noradrenaline. Rb+, but not Li+, had the capacity to replace internal K+. Two explanations are proposed for this effect of internal K+: (a) creation of a K+ diffusion potential (inside negative) provides a driving force for inward transport, and/or (b) K+ increases the turnover rate by formation of a highly mobile potassium-carrier complex. A hypothetical scheme for the transport of noradrenaline is presented.     

Maintaining osmotic balance with an aglomerular kidney

The gulf toadfish, Opsanus beta, is a marine teleost fish with an aglomerular kidney that is highly specialized to conserve water. Despite this adaptation, toadfish have the ability to survive when in dilute hypoosmotic seawater environments. The objectives of this study were to determine the joint role of the kidney and intestine in maintaining osmotic and ionic balance and to investigate whether toadfish take advantage of their urea production ability and use urea as an osmolyte. Toadfish were gradually acclimated to different salinities (0.5, 2.5, 5, 10, 15, 22, 33, 50 and 70 ppt (1.5%, 7.5%, 15%, 30%, 45%, 67%, 100%, 151% and 212% seawater)) and muscle tissue, urine, blood and intestinal fluids were analyzed for ion and in some cases urea concentration. The renal and intestinal ionoregulatory processes of toadfish responded to changes in salinity and when gradually acclimated, toadfish maintain a relatively constant plasma osmolality at environmental salinities of 5 to 50 ppt. However, at salinities lower (2.5 ppt) or higher (70 ppt) than this range, a significant deviation from resting plasma and urine osmolality as well as changes in muscle water content was measured, suggesting osmoregulatory difficulties at these salinities. The renal system compensates for dilute seawater by reducing Na+ reabsorption by the bladder, which allowed excess water to be excreted. In the case of hypersalinity, Na+ reabsorption was increased, which resulted in a conservation of water and the concentration of Mg2+, Cl-, SO(4)2- and urea. A similar pattern was observed within the gastrointestinal system. Notably, Mg2+, HCO3- and SO4(2-) were the dominant ions in the intestinal fluid under control and hypersaline conditions due to the absorption of Na+, Cl- and water. When exposed to dilute seawater conditions, the absorption of Na+ was greatly reduced which likely increased water elimination. As a result of decreased environmental levels and a reduction in drinking rate, Mg2+ and SO4(2-) in intestinal fluids under hypoosmotic conditions were greatly reduced. While urea did play a minor role in renal osmoregulation, toadfish appear to preferentially regulate Na+ and to some extend Cl- in urine and intestinal fluids.     

Swelling and potassium uptake in cultured astrocytes

The intracellular water content of astrocytes in primary cultures shows a biphasic swelling pattern on exposure to various increased external K+ concentrations over the range of 1.5-100 mM. The two phases (physiological, 1.5-12 mM K+; pathological, 25-100 mM K+) are based on two different mechanisms. Both can be blocked by low Cl- solutions and involve intensive net uptake of K+. However, the physiological phase consists of the activation of a KCl + NaCl carrier, while the Na+ in turn is pumped out by Na+-K+ ATPase, with a resultant net accumulation of KCl. At pathological K+ concentrations the KCl + NaCl carrier is less active because the Na+ driving force, its energy source, is reduced (owing to depolarization by K+). However, the Donnan equilibrium across the cell membrane is heavily disturbed, which leads to passive KCl accumulation. The results suggest that volume changes in cultured glial cells during exposure to high K+ should be taken into consideration since they disguise K+ accumulation when only ion activity is measured.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.     

Socially-induced changes in sodium regulation affect the uptake of water-borne copper and silver in the rainbow trout,Oncorhynchus mykiss

Subordinate fish take up more copper during water-borne exposure than dominant fish and consequently display higher tissue burdens. The present study demonstrated a similar effect of social status on water-borne silver uptake. We evaluated whether differences in copper and silver accumulation between individuals could be due to differences in metabolic rate, internal concentrations of cortisol or sodium uptake rates. In the absence of social interaction, experimentally increased metabolic rates (via moderate exercise) and elevated whole body cortisol concentrations (via feeding of a cortisol-spiked diet) did not result in increased metal uptake. However, elimination of the difference in sodium uptake rates between dominant and subordinate fish by exposing them to a saturating level of water-borne sodium (50 mM) resulted in an elimination of copper uptake differences. No significant differences in sodium and silver uptake rates were seen between dominant and subordinate fish exposed to elevated silver concentrations. Therefore, it appears that socially-mediated differences in copper and silver accumulation are a result of differences in sodium uptake rates as both silver and copper are known to cross the gill epithelia via sodium transport pathways.     

Effects of copper on the acute cortisol response and associated physiology in rainbow trout

The aim of this study was to determine the effects of chronic waterborne copper (Cu) exposure on the acute stress-induced cortisol response and associated physiological consequences in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 30 μg Cu/L in moderately hard water (120 mg/L as CaCO(3)) for 40 days, following which time the acute cortisol response was examined with a series of stressors. At 40 days, a 65% increase in Cu was observed in the gill, but no accumulation was observed in the liver, brain or head kidney. Stressors such as air exposure or confinement did not elicit an increase in circulating cortisol levels for Cu-exposed fish, in contrast to controls. However, this inhibitory effect on the acute cortisol response appeared to have few implications on the ability of Cu-exposed fish to maintain ion and carbohydrate homeostasis. For example, plasma Na(+), Ca(2+) and glucose levels as well as hepatic glycogen levels were the same post-stress in control and Cu-exposed fish. Trout were also challenged with exposure to 50% seawater for 48 h, where Cu-exposed trout maintained plasma Na(+), glucose and hepatic glycogen levels. However, Cu-exposed fish experienced decreased plasma K(+) levels throughout the Cu exposure and stress tests. In conclusion, chronic Cu exposure resulted in the abolition of an acute cortisol response post-stress. There was no Cu accumulation in the hypothalamus-pituitary-interrenal axis (HPI axis) suggesting this was not a direct toxic effect of Cu on the cortisol regulatory pathway. However, the lack of an acute cortisol response in Cu-exposed fish did not impair the ability of the fish to maintain ion and carbohydrate homeostasis. This effect on cortisol may be a strategy to reduce costs during the chronic stress of Cu exposure, and not endocrine disruption as a result of toxic injury.     

Concentration of MgSO4 in the intestinal lumen of Opsanus beta limits osmoregulation in response to acute hypersalinity stress

Marine teleosts constantly lose water to their surrounding environment, a problem exacerbated in fish exposed to salinity higher than normal seawater. Some fish undergo hypersaline exposures in their natural environments, such as short- and long-term increases in salinity occurring in small tidal pools and other isolated basins, lakes, or entire estuaries. Regardless of the degree of hypersalinity in the ambient water, intestinal absorption of monovalent ions drives water uptake to compensate for water loss, concentrating impermeable MgSO(4) in the lumen. This study considers the potential of luminal [MgSO(4)] to limit intestinal water absorption, and therefore osmoregulation, in hypersalinity. The overall tolerance and physiological response of toadfish (Opsanus beta) to hypersalinity exposure were examined. In vivo, fish in hypersaline waters containing artificially low [MgSO(4)] displayed significantly lower osmolality in both plasma and intestinal fluids, and increased survival at 85 parts per thousand, indicating improved osmoregulatory ability than in fish exposed to hypersalinity with ionic ratios similar to naturally occurring ratios. Intestinal sac preparations revealed that in addition to the osmotic pressure difference across the epithelium, the luminal ionic composition influenced the absorption of Na(+), Cl(-), and water. Hypersalinity exposure increased urine flow rates in fish fitted with ureteral catheters regardless of ionic composition of the ambient seawater, but it had no effect on urine osmolality or pH. Overall, concentrated MgSO(4) within the intestinal lumen, rather than renal or branchial factors, is the primary limitation for osmoregulation by toadfish in hypersaline environments.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Water chloride provides partial protection during chronic exposure to waterborne silver in rainbow trout (Oncorhynchus mykiss) embryos and larvae

Rainbow trout embryos and larvae were continuously exposed (at 12.5 degrees C) to waterborne silver in a flow-through setup, from fertilization to swim-up, at nominal silver concentrations of 0, 0.1, or 1.0 microg/L total silver (as AgNO(3)) at three different water Cl(-) levels (30, 300, and 3,000 microM, added as KCl). Exposures were conducted in synthetic soft water (hardness 20 mg CaCO(3)/L generated from reconstituted reverse osmosis freshwater). Continuous exposure to 1.0 microg/L total silver for 58 d at 30 microM water Cl(-) resulted in a pronounced ionoregulatory disturbance (as indicated by a reduction in whole body Na(+),K(+)-ATPase activity, unidirectional Na(+) uptake [Jin Na(+)], and whole body Na(+) and Cl(-) levels) and a reduction in extractable protein and wet weight. Thus, the mechanism of chronic silver toxicity appears to be similar to that observed during acute silver exposure in juvenile and adult fish, specifically an ionoregulatory disturbance. Higher water Cl(-) levels (300 and 3,000 microM Cl(-)) offered some degree of protection from the ionoregulatory disturbance, with only minor protective effects in terms of mortality. The protective effects of water Cl(-) on the toxicity of silver (as AgNO(3)) appear to be far less during chronic than during acute exposure. Mortality and larval Na(+) concentration, Jin Na(+), and Na(+),K(+)-ATPase activity all appear to be correlated with silver body burden and calculated water Ag(+) during chronic silver exposure. Thus, there appears to be potential to model chronic toxicity but not simply by recalibration of an acute model. A chronic model must be based on real chronic data because the protective effects of various ligands appear to be quantitatively very different from those in the acute situation.     

Regulation of salt gland, gut and kidney interactions

Marine birds can drink seawater because their cephalic 'salt' glands secrete a sodium chloride (NaCl) solution more concentrated than seawater. Salt gland secretion generates osmotically free water that sustains their other physiological processes. Acclimation to saline induces interstitial water and Na move into cells. When the bird drinks seawater, Na enters the plasma from the gut and plasma osmolality (Osm(pl)) increases. This induces water to move out cells expanding the extracellular fluid volume (ECFV). Both increases in Osm(pl) and ECFV stimulate salt gland secretion. The augmented intracellular fluid content should allow more rapid expansion of ECFV in response to elevated Osm(pl) and facilitate activation of salt gland secretion. To fully utilize the potential of the salt glands, intestinally absorbed NaCl must be reabsorbed by the kidneys. Thus, Na uptake at gut and renal levels may constrain extrarenal NaCl secretion. High NaCl intake elevates plasma aldosterone concentration of Pekin ducks and aldosterone stimulates intestinal and renal water and sodium uptake. High NaCl intake induces lengthening of the small intestine of adult Mallards, especially males. High NaCl intake has little effect on glomerular filtration rate or tubular sodium Na uptake of birds with competent salt glands. Relative to body mass, kidney mass and glomerular filtration rate (GFR) are greater in birds with salt glands than in birds that do not have them. Birds with salt glands do not change GFR, when they drink saline. Thus, their renal filtrate contains excess Na that is, in some species, almost completely renally reabsorbed and excreted in a more concentrated salt gland secretion. Na reabsorption by kidneys of other species, like mallards is less complete and their salt glands make less concentrated secretion. Such species may reflux urine into the hindgut, where additional Na may also be reabsorbed for extrarenal secretion. During exposure to saline, marine birds maintain elevated aldosterone levels despite high Na intake. Marine birds are excellent examples of physiological plasticity.     

Electrogenicity of the Na+-ATPase from the marine microalga Tetraselmis (Platymonas) viridis and associated H+ countertransport

Sodium accumulation by the Na+-ATPase in the plasma membrane (PM) vesicles isolated from the marine alga Tetraselmis (Platymonas) viridis was shown to be accompanied by deltapsi generation across the vesicle membrane (positive inside) and H+ efflux from the vesicle lumen. Na+ accumulation was assayed with 22Na+; deltapsi generation was detected by recording absorption changes of oxonol VI; H+ efflux was monitored as an increase in fluorescence intensity of the pH indicator pyranine loaded into the vesicles. Both ATP-dependent Na+ uptake and H+ ejection were increased by the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CICCP) while deltapsi was collapsed. The lipophilic anion tetraphenylboron ion (TPB-) inhibited H+ ejection from the vesicles and abolished deltapsi. Based on the effects of CICCP and TPB- on H+ ejection and deltapsi generation, the conclusion was drawn that H+ countertransport observed during Na+-ATPase operation is a secondary event energized by the electric potential which is generated in the course of Na+ translocation across the vesicle membrane. Increasing Na+ concentrations stimulated H+ efflux and caused the decrease in the deltapsi observed, thus indicating that Na+ is likely a factor controlling H+ permeability of the vesicle membrane.     

Exposure to fluctuating salinity enhances free amino acid accumulation inTigriopus californicus (Copepoda)

Summary Intracellular concentrations of free amino acids (FAA) in the intertidal copepodTigriopus californicus increase in response to hyperosmotic stress and decrease in response to hypo-osmotic stress. The purpose of this study was to determine if exposure to repeated bouts of osmotic stress resulted in changes in FAA accumulation or the degree of FAA retention in subsequent episodes. Five groups ofT. californicus were exposed for 22 days to a fluctuating salinity regime which consisted of 24 h at 100% seawater followed by 24 h at either 90, 80, 70, 60 or 50% seawater (11 cycles). After the tenth exposure to 100% seawater, individuals from each treatment group were analyzed for alanine and proline concentration. Alanine and proline accumulation generally increased in proportion to the osmotic stress up to 60–100% seawater — additional osmotic stress failed to increase total accumulation. Prior exposure to fluctuating salinity increased the extent of alanine and proline retention observed upon transfer to a hypo-osmotic medium. The treatment group which had experienced the most extreme fluctuation (50–100% seawater) retained alanine and proline levels approximately 10- and 20-fold higher, respectively, than controls. A less severe salinity fluctuation was required to elicit this response for alanine (90–100% seawater) than for proline (60–100% seawater). Previous exposure to fluctuating salinity also resulted in increased alanine and proline accumulation in subsequent episodes of hyperosmotic stress. 24 h after transfer from 50 to 100% seawater, alanine and proline levels in the conditioned copepods were approximately 3- and 7-fold higher, respectively, than in copepods which had not been cycled. This facilitation in alanine and proline accumulation occurred after 10 and 11 cycles, respectively. Of the increased accumulation in alanine and proline, 7.0% and 22.5%, respectively, could be accounted for by the higher degree of FAA retention while under hypo-osmotic conditions.Abbreviation FAA free amino acids     

Changes in the levels of chloride cells and (Na+ + K+)-dependent ATPase in the gills of yellow and silver eels adapting to seawater.

Changes were measured in the numbers of chloride cells and the levels of (Na+ + K+)-DEPENDENT ATPase in the gills of immature, yellow eels and mature, silver eels during adaptation from freshwater to seawater. The percentage of chloride cells in yellow eels more than doubled after six days in seawater; at this time the specific activity and concentration of (Na+ + K+)-dependent ATPase in gills start to increase in parallel to reach maxima after two weeks that are 2.5 times the starting values. It is concluded that adaptation of yellow eels to seawater involves an increase in the numbers of chloride cells in gills as well as an increased amount of (Na+ + K+)-dependent ATPase per chloride cell. Mature silver eels in freshwater had essentially the same numbers of chloride cells and the same specific activity of the enzyme in the gills as yellow eels fully adapted to seawater. Transferring silver eels to seawater did not alter the percentage of chloride cells in gills although the level of (Na+ + K+)-dependent ATPase and its specific activity increased slightly. Thus, although the silver eel is better prepared for life in seawater than the yellow eel, it still has to attain an increased level of (Na+ + K+)-dependent ATPase in its chloride cells to be fully adapted to seawater.     

The physiological basis for altered Na+ and Cl- movements across the gills of rainbow trout (Oncorhynchus mykiss) in alkaline (pH = 9.5) water

To test the hypothesis that internal ion imbalances at high pH are caused by altered branchial ion transporting capacity and permeability, radiotracers (24Na+ and 36Cl-) were used to measure ion movements across the gills of intact rainbow trout (Oncorhynchus mykiss) during 3 d exposure to pH 9.5. At control pH (pH 8.0), the trout were in net ion balance, but by 8 h at high pH, 60%-70% reductions in Cl- influx (JClin) and Na+ influx (JNain) led to net Cl- and Na+ losses of -200 micromol kg-1 h-1. Outflux (diffusive efflux plus renal ion losses) was not initially altered. By 72 h, net Cl- balance was reestablished because of a restoration of JClin. Although JNain remained 50% lower at this time, counterbalancing reductions in Na+ outflux restored net Na+ balance. One-substrate ion-uptake kinetics analyses indicated that reduced ion influx after 8 h at pH 9.5 was caused by 50% decreases in Cl- and Na+ maximal transport rates (JClmax, JNamax), likely reflecting decreased numbers of functional transport sites. Two-substrate kinetic analyses indicated that reduced internal HCO-3 and H+ supply for respective branchial Cl-/base and Na+/acid transport systems also contributed to lower JClin and, to a lesser extent, lower JNain at pH 9.5. Recovery of JClmax after 3 d accounted for restoration of Cl- balance and likely reflected increased numbers of transport sites. In contrast, JNamax remained 33% lower after 3 d, but a lower affinity of the gills for Na+ (fourfold greater KNam) accounted for the chronic reduction in Na+ influx at pH 9.5. Thus, reestablishment of Cl- uptake capacity and counterbalancing reductions in Na+ outflux allows rainbow trout to reestablish net ion balance in alkaline waters.