Parathyroid hormone-related protein regulates intestinal calcium transport in sea bream (Sparus auratus)

Parathyroid hormone-related protein (PTHrP) is a factor associated with normal development and physiology of the nervous, cardiovascular, immune, reproductive, and musculoskeletal systems in higher vertebrates. It also stimulates whole body calcium uptake in sea bream (Sparus auratus) larvae with an estimated 60% coming from intestinal uptake in seawater. The present study investigated the role of PTHrP in the intestinal calcium transport in the sea bream in vitro. Unidirectional mucosal-to-serosal and serosal-to-mucosal 45Ca fluxes were measured in vitro in duodenum, hindgut, and rectum mounted in Ussing chambers. In symmetric conditions with the same saline, bathing apical and basolateral sides of the preparation addition of piscine PTHrP 1-34 (6 nM) to the serosal surface resulted in an increase in mucosal to serosal calcium fluxes in duodenum and hindgut and a reduction in serosal to mucosal in the rectum, indicating that different mechanisms are responsive to PTHrP along the intestine. In control asymmetric conditions, with serosal normal and mucosal bathed with a saline similar in composition to the intestinal fluid, there was a net increase in calcium uptake in all regions. The addition of 6 nM PTHrP 1-34 increased net calcium uptake two- to threefold in all regions. The stimulatory effect of PTHrP on net intestinal calcium absorption is consistent with a hypercalcemic role for the hormone. The results support the view that PTHrP, alone or in conjunction with recently identified PTH-like peptides, counteracts in vivo the hypocalcemic effects of stanniocalcin.     

Parathyroid hormone-related protein: a calcium regulatory factor in sea bream (Sparus aurata L.) larvae

The effects of an N-terminal peptide (amino acids 1-38) of Fugu parathyroid hormone-related protein (PTHrP 1-38) on calcium regulation of larval sea bream were investigated in seawater (36 per thousand) and after transfer to dilute seawater (12 per thousand). Exposure to PTHrP 1-38 evoked a 1.5-fold increase in calcium influx in both full-strength and dilute seawater. Calcium influx in dilute seawater-adapted larvae was roughly one-half that observed in full-strength seawater controls. PTHrP 1-38 also reduced drinking of fish in seawater but, at all concentrations tested, was without effect in dilute seawater. The amount of water imbibed was 55% lower in dilute seawater than in seawater. PTHrP 1-38 exposure affected the calcium influx route: the main contribution of calcium uptake shifted from intestinal absorption to extraintestinal uptake, probably by the induction of a dose-dependent increase in branchial (active) transport. Moreover, seawater-adapted fish exposed to 1 nM and 10 mM PTHrP 1-38 experienced a 2.5-fold reduction in overall calcium efflux. Overall, the calciotropic action of PTHrP 1-38 resulted in a dose-dependent increase in net calcium balance.     

A PTH/PTHrP receptor antagonist blocks the hypercalcemic response to estradiol-17beta

Estradiol (E(2)) increases circulating calcium and phosphate levels in fish, thus acting as a hypercalcemic and hyperphosphatemic factor during periods of high calcium requirements, such as during vitellogenesis. Since parathyroid hormone (PTH)-related protein (PTHrP) has been shown to be calciotropic in fish, we hypothesized that the two hormones could be mediating the same process. Sea bream (Sparus auratus) juveniles receiving a single intraperitoneal injection of piscine PTHrP(1-34) showed an elevation in calcium plasma levels within 24 h. In contrast, injections of the PTH/PTHrP receptor antagonist PTHrP(7-34) decreased circulating levels of calcium in the same period. Intraperitoneal implants of estradiol-17beta (E(2); 10 microg/g) evoked significant increases of circulating plasma levels of calcium and phosphorus and a sustained increases of circulating plasma levels of PTHrP. However, a combined treatment of E(2) and PTHrP(7-34) evoked a markedly lower calcium response compared with E(2) alone. We conclude that PTHrP or a related peptide that binds the PTH/PTHrP receptor mediates, at least in part, the hypercalcemic effect of E(2) in calcium and phosphate balance in fish.     

The effects of hypercapnia on branchial and renal calcium fluxes in the rainbow trout (Oncorhynchus mykiss )

Whole body calcium influx, branchial calcium efflux, and renal Ca²⁺ excretion were measured in rainbow trout (Oncorhynchus mykiss) exposed to hypercapnia. These experiments were performed to assess the potential impact on Ca²⁺ balance of the changes in gill morphology known to accompany respiratory acidosis in this species. After 48 h of hypercapnia, gill filamental chloride cell fractional area was significantly reduced. Despite this reduction and the presumed involvement of the chloride cell in calcium influx, whole body calcium influx was increased after 12 h of hypercapnia and remained elevated for 48 h. Branchial calcium efflux was unaltered during hypercapnia exposure, whereas renal Ca²⁺ excretion was elevated over preflux values only at 6 h of hypercapnia. Measurement of the kinetics of whole body calcium influx after 48 h of hypercapnia revealed a significant increase in the maximal uptake rate of Ca²⁺, yet the affinity constant of Ca²⁺ uptake was unaffected. Measurements of high-affinity Ca²⁺ -ATPase activities and ATP-dependent Ca²⁺ transport of gill basolateral membrane vesicles revealed that the ATP-dependent Ca²⁺ extrusion mechanism of the gills was not affected by hypercapnia. The results of the present study clearly show that the reduced chloride cell surface area that accompanies hypercapnia in trout does not impair calcium homeostasis. Although adjustments to the basolateral membrane high affinity Ca²⁺ transporter do not appear to play a role, the mechanism(s) underlying the maintenance of calcium homeostasis under hypercapnic conditions are unresolved. Accepted: 1 July 1996     

Dietary phosphorus affected growth performance,body composition,antioxidant status and total P discharge of young hybrid sturgeon (♀Huso huso × ♂Acipenser schrenckii) in winter months

An 8‐week feeding trial was carried out using young hybrid sturgeon (♀Huso huso × ♂Acipenser schrenckii) to study the effects of dietary phosphorus (P) on growth performance, body composition, liver and serum antioxidant status as well as the effluent P content in the wintertime. Four puffed pellet diets were formulated to contain graded total P levels at 0.63, 1.15, 1.85, and 2.12%, respectively. Triplicate groups of hybrid sturgeon (494.21 ± 18.63 g) were reared in concrete ponds (2.0 × 1.2 × 1.4 m), and fed one of the four diets for 8 weeks. Weight gain and feeding rate significantly decreased with increasing dietary P content, and fish fed the 2.12% P diet had negative growth. With the increase in dietary P, whole body ash and P content significantly increased. There was no inverse relationship between the whole body lipid content and dietary P level in young hybrid sturgeon. Hybrid sturgeon liver exhibited higher superoxide dismutase, catalase and glutathione peroxidase activities in the 0.63 and 1.15% dietary P treatment groups than those in the other two groups. Serum alkaline phosphatase activity increased significantly with increasing dietary P. Discharge of P into the pond increased significantly with increasing dietary P. In conclusion, a dietary P content between 0.63 and 1.15% helped young hybrid sturgeon to activate their antioxidant defense system and eradicate the free radicals, in order to reduce the antioxidant damage, while still keeping total P content in the pond at a relatively low level during the winter months.     

Effects of epinephrine on branchial and renal calcium handling in the rainbow trout

Acute exposure of rainbow trout (Salmo gairdneri) to low external calcium (25 microM) caused an immediate but transient increase in plasma epinephrine concentration that may have been related to a concomitant depression of blood pH. Intra-arterial infusion of epinephrine at normal ambient calcium levels (0.35 mM) for 4 h caused circulating levels of epinephrine to rise from 2.9 X 10(-9) to 8.0 X 10(-8) M but did not affect norepinephrine levels, or branchial unidirectional calcium fluxes. Active (ATP-dependent) calcium transport across basolateral plasma membranes prepared from gill epithelial cells was not affected by pretreatment of fish with epinephrine or by direct application of epinephrine or cAMP, in vitro. Epinephrine infusion elevated urine flow rate, decreased urine pH, and increased urine phosphate levels significantly. Net renal calcium efflux increased significantly as a result of the increased urine flow rate. It is concluded that epinephrine does not stimulate branchial calcium uptake or renal conservation of calcium in rainbow trout at normal external calcium levels and therefore we cautiously suggest that epinephrine is unlikely to be involved in calcium balance during periods of exposure to low external calcium. Instead, epinephrine may play a role in compensating the acid-base disturbances and the increased branchial water influx that are associated with exposure to low ambient calcium.     

The Effect of Calcium on Non-heme Iron Uptake,Efflux, and Transport in Intestinal-like Epithelial Cells (Caco-2 Cells)

It has been suggested that calcium inhibits the absorption of dietary iron by directly affecting enterocytes. However, it is not clear if this effect is due to a decreased uptake of iron or its efflux from enterocytes. We studied the effect of calcium on the uptake, efflux, and net absorption of non-heme iron using the intestinal-like epithelial cell line Caco-2 as an in vitro model. Caco-2 cells were incubated for 60 min in a buffer supplemented with non-heme iron (as sulfate) and calcium to achieve calcium to iron molar ratios ranging from 50:1 to 1,000:1. The uptake, efflux, and net absorption of non-heme iron were calculated by following a radioisotope tracer of ⁵⁵Fe that had been added to the buffer. Administration of calcium and iron at molar ratios between 500 and 1,000:1 increased the uptake of non-heme iron and decreased efflux. Calcium did not have an effect on the net absorption of non-heme iron. At typical supplementary doses for calcium and non-heme iron, calcium may not have an effect on the absorption of non-heme iron. The effect of higher calcium to iron molar ratios on the efflux of non-heme iron may be large enough to explain results from human studies.     

Net uptake of chloride across the posterior gills of the Chinese crab (Eriocheir sinensis)

Two methods are commonly used for the determination of transbranchial net fluxes of Na+ and Cl-: direct analysis of changes in ion concentrations in the external medium using flame spectrophotometry or titration (net flux method), and measurement of unidirectional ion fluxes by means of radioactive tracers (tracer method). When we applied both methods in the same preparation, the isolated perfused posterior gill of freshwater-acclimated Eriocheir sinensis, to determine net fluxes of Cl-, the results differed substantially. In artificial fresh water (AFW) containing NaCl, the net flux method yielded a net uptake, but the tracer method showed a net efflux of Cl-. The net uptake of Cl- was abolished in Na(+)-free AFW indicating that Cl- uptake is coupled with the uptake of Na+. Applying the tracer method, net efflux of Cl- remained almost unchanged in Na(+)-free AFW. This suggests the opposite mechanism, i.e. uncoupled uptake of Na+ and Cl-. The discrepancy in the results obviously depends on the method employed. Since the data obtained with the net flux method explain the osmoregulatory performance of crabs living in fresh water, we consider this method as appropriate for determining net transbranchial ion fluxes.     

Uteroplacental restriction in the rat impairs fetal growth in association with alterations in placental growth factors including PTHrP

During pregnancy, parathyroid hormone-related protein (PTHrP) is one of many growth factors that play important roles to promote fetal growth and development, including stimulation of placental calcium transport. Angiotensin II, acting through the AT(1a) receptor, is also known to promote placental growth. We examined the effects of bilateral uterine artery and vein ligation (restriction), which mimics placental insufficiency in humans, on growth, intrauterine PTHrP, placental AT(1a), and pup calcium. Growth restriction was surgically induced on day 18 of pregnancy in Wistar-Kyoto female rats by uterine vessel ligation. Uteroplacental insufficiency reduced fetal body weight by 15% and litter size (P < 0.001) compared with the control rats with no effect on placental weight or amniotic fluid volume. Uteroplacental insufficiency reduced placental PTHrP content by 46%, with increases in PTHrP (by 2.6-fold), parathyroid hormone (PTH)/PTHrP receptor (by 11.6-fold), and AT(1a) (by 1.7-fold) relative mRNA in placenta following restriction compared with results in control (P < 0.05). There were no alterations in uterine PTHrP and PTH/PTHrP receptor mRNA expression. Maternal and fetal plasma PTHrP and calcium concentrations were unchanged. Although fetal total body calcium was not altered, placental restriction altered perinatal calcium homeostasis, as evidenced by lower pup total body calcium after birth (P < 0.05). The increased uterine and amniotic fluid PTHrP (P < 0.05) may be an attempt to compensate for the induced impaired placental function. The present study demonstrates that uteroplacental insufficiency alters intrauterine PTHrP, placental AT(1a) expression, and perinatal calcium in association with a reduction in fetal growth. Uteroplacental insufficiency may provide an important model for exploring the early origins of adult diseases.     

Cortisol stimulates calcium transport across cultured gill epithelia from freshwater rainbow trout

Summary The effect of cortisol on calcium (Ca²⁺) transport across cultured rainbow trout gill epithelia composed of both pavement cells (PVCs) and mitochondria-rich cells (MRCs) was examined. Under symmetrical culture conditions (L15 media apical/L15 media basolateral), cortisol had subtle effects on gill epithelial preparations. Both control and cortisol treated epithelia exhibited Ca²⁺ influx and efflux rates (measured radioisotopically using ⁴⁵Ca) that were approximately balanced, with a slight inwardly directed net Ca²⁺ flux. Ussing flux ratio analysis indicated active Ca²⁺ transport in the inward direction across epithelia bathed symmetrically regardless of hormone treatment. In contrast, under asymmetrical conditions (freshwater apical/L15 media basolateral) control epithelia exhibited active Ca²⁺ transport in the outward direction (basolateral to apical) throughout experiments conducted over a 24-h period, whereas cortisol-treated preparations exhibited active transport in the inward direction (apical to basolateral) during the early stages of an asymmetrical culture period (e.g., T0–6 h) and passive transport during the later stages (e.g., T18–24 h). When soft freshwater (with tenfold lower [Ca²⁺]) was used for asymmetrical culture instead of freshwater, control epithelia developed outwardly directed active Ca²⁺ transport properties, whereas cortisol-treated preparations did not. The results of this study support a hypercalcemic role for cortisol in rainbow trout and demonstrate that treating cultured gill epithelia composed of both PVCs and MRCs with cortisol can stimulate active Ca²⁺ uptake under circumstances that more closely resemble natural conditions for fish gills (i.e., freshwater bathing the apical side of the epithelium).     

Effects of Low Root Temperature on Ion Uptake and Ion Translocation in Wheat

Roots of wheat seedlings (Triticum aestivum L. cv. Weibulls Starke) were cooled (+1°C) for 24 h while the shoots were kept at 25°C. The treatment induced an increased water deficit in the leaves. Fresh weight, dry weight, and the uptake and distribution of potassium and calcium were measured before and after cooling. Growth, measured both as fresh weight and dry weight increase, was reduced during the cold treatment. Afterwards (at 20°C), growth recovered to nearly pre-stress rates. Analysis of the potassium fluxes in and out of the roots by ⁸⁶Rb techniques showed that influx, and to a lesser extent efflux, were inhibited at low temperature. The result was a net potassium uptake rate of one-third that of unstressed plants. After the cooling period the potassium influx increased to the rate of control plants. The potassium efflux increased to one and one-half times the rate of unstressed wheat so that net uptake was negative. The increase in potassium efflux was explained by a higher permeability of the root cell membranes after cooling. The net uptake of calcium was reduced to one-third by root cooling. Contrary to potassium uptake, calcium uptake increased under post-stress conditions, partly due to a low efflux rate. During root cooling there was a redistribution of dry matter from the leaves down towards the lower part of the shoot. Afterwards the original distribution of dry matter was reestablished. The net flow of potassium and calcium followed a similar pattern as dry matter, suggesting a growth-regulated flow.     

Calcium balance in crustaceans: nutritional aspects of physiological regulation

Calcium homeostasis in crustaceans is influenced by their natural molting cycle that periodically requires replacement of the calcified exoskeleton in order for growth to occur. Whole body Ca balance transitions from intermolt (zero net flux) to premolt (net efflux) and postmolt (net influx at the rate of 2 mmol kg(-1)h(-1)). As such, molting provides a convenient model to study up- and down-regulation of epithelial Ca transporting proteins (such as Ca pumps and exchangers), the genes that encode them, and the steroid hormone (ecdysone) that putatively regulates the genes. Species residing in either freshwater or in terrestrial environments are more limited in their Ca availability than are marine species. Further the advance towards terrestriality is accompanied by decreased reliance upon branchial Ca uptake and increased reliance upon digestive uptake. This review will correlate Ca handling strategies with environment in semi-terrestrial and terrestrial crabs through examining environmental sources of Ca uptake. Ca homeostasis will also be discussed at the whole animal level, cellular, subcellular and molecular levels of regulation.     

Mechanisms behind Pb-induced disruption of Na+ and Cl- balance in rainbow trout (Oncorhynchus mykiss)

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.     

Some effects of adrenaline on anion transport and nitrite-induced methaemoglobin formation in the rainbow trout (Salmo gairdneri Richardson)

The effects of adrenaline on branchial anion transport and nitrite-induced methaemoglobinaemia have been investigated in rainbow trout. Nitrite uptake and efflux results suggest that adrenaline effects a net anion efflux principally by stimulation of the unidirectional branchial anion efflux. In oxygenated whole blood nitrite-induced methaemogloblin was significantly reduced in the presence of adrenaline. The physiological and environmental consequences of nitrite-induced stress are discussed.     

Effects of continuous infusion of parathyroid hormone and parathyroid hormone-related peptide on rat bone in vivo: comparative study by histomorphometry

We have investigated the actions of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) on the bones of parathyroidectomized (PTX) rats by histomorphometric analysis. Miniosmotic pumps filled with either human PTH (hPTH)(1–34), hPTHrP(1–34) or vehicle were subcutaneously implanted on the backs of the rats. The peptides were continuously infused for 6 days at a rate of 15 nmole/kg/day. PTH and PTHrP exhibited similar hypercalcemic and hypophosphatemic actions on these PTX rats. No significant differences were noted in bone weight or calcium and phosphorus contents of the ashed bone among the 3 groups. By quantitative histomorphometric analysis, hPTH(1–34) and hPTHrP(1–34) were found similarly to enhance both bone formation and resorption. Peritrabecular fibrosis was observed only in the PTH-infused animals. PTHrP thus mimics the actions of PTH, but is not as effective in promoting mesenchymal cell proliferation along the bone trabeculae.     

Effect of secondary metabolites associated with anaerobic soil conditions on ion fluxes and electrophysiology in barley roots

The effects of secondary metabolites produced by waterlogged soils on net K(+), H(+), and Ca(2+) fluxes were studied in the mature zone of roots of two barley (Hordeum vulgare) cultivars contrasting in their waterlogging (WL) tolerance using the noninvasive microelectrode ion flux measuring technique. In WL-sensitive variety 'Naso Nijo', all three lower monocarboxylic acids (formic, acetic, and propionic acids) and three phenolic acids (benzoic, 2-hydroxybenzoic, 4-hydroxybenzoic acids) caused a substantial shift toward steady K(+) efflux, accompanied by an immediate net influx of H(+). Detrimental effects of secondary metabolites on K(+) homeostasis in root cells were absent in WL-tolerant 'TX' variety. Root treatment with Mn(2+) caused only a temporary K(+) loss that returned to the initial level 10 min after treatment. Phenolic acids slightly increased Ca(2+) influx immediately after treatment, while other metabolites tested resulted in transient Ca(2+) efflux from the root. In the long-term (24 h) treatment, all metabolites tested significantly reduced K(+) uptake and the adverse effects of phenolic acids were smaller than for monocarboxylic acids and Mn(2+). Treatment with monocarboxylic acids for 24 h shifted H(+) from net efflux to net influx, while all three phenolic acids did not cause significant effects compared with the control. Based on results of pharmacological experiments and membrane potential measurements, a model explaining the effects of secondary metabolites on membrane transport activity is proposed. We also suggest that plant tolerance to these secondary metabolites could be considered a useful trait in breeding programs.     

Effect of extracellular volume expansion on erythrocyte sodium transport in rats.

The effects of extracellular volume expansion (EVE) on the major sodium transport systems and sodium and potassium contents in rat erythrocytes have been examined in the present study. Study has been performed in anesthetized Wistar rat weighing about 300 g. Acute extracellular volume expansion (EVE) was induced by a constant intravenous saline infusion (3% body wt, 3 hours). Rats anaesthetized and catheterized but not expanded were used as controls. Arterial blood samples from control and expanded rats were obtained at the same time, and assayed immediately. Intracellular sodium and potassium concentration and ouabain sensitive (Na(+)-K(+)-pump) and bumetanide sensitive (Na(+)-K(+)-cotransport system) outward Na+ fluxes in erythrocytes were measured. The effect of plasma on erythrocyte transport was also analyzed by measuring 86Rb uptake. Neither of two plasma cations (Na+ and K+) were modified by the EVE. Also intracellular Na+ and K+ levels remained unvariable. Total Na+ efflux was not modified by EVE, but pump-mediated Na+ efflux was smaller after than before EVE. The ouabain-inhibible Na+ efflux rate constant decreased after EVE (from 687 +/- 81 to 525 +/- 29 h-1 x 10(-3); P less than 0.05). Both Na(+)-K(+)cotransport-mediated Na+ efflux and passive permeability increased significantly after EVE. The incubation with plasma from saline-infused animals induced a significant decrease in Rb uptake rate constant, that was not observed after incubation with plasma from non-expanded rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low unidirectional sodium influx into root cells restricts net sodium accumulation in Thellungiella halophila, a salt-tolerant relative of Arabidopsis thaliana

Thellungiella halophila is a useful model species for research into plant salt tolerance. It is closely related to Arabidopsis thaliana, but shows considerably higher salt tolerance. Comparative analysis of ion homeostasis in the two species allows the identification of ion transport pathways that are critical for salt tolerance and provides the basis for future studies into their molecular features. Previous studies indicated that salt tolerance in T. halophila is accompanied by low accumulation of Na in the leaves. Kinetic analysis of net ion uptake over three days confirmed lower Na uptake and K loss in T. halophila compared with A. thaliana. Differential net Na uptake rates were still apparent after 6 weeks of salt treatment. To assess the contribution of unidirectional Na fluxes to net Na uptake, kinetic studies of (22)Na fluxes were carried out in both species. The results show that unidirectional root Na influx is significantly lower in salt-grown T. halophila plants than in A. thaliana exposed to the same level of salinity (100 mM). Quantitative comparison of unidirectional influx and net Na accumulation suggests that both species operate efficient Na efflux, which partly compensates for Na influx. Kinetic analysis of (22)Na efflux indicated higher root Na efflux in A. thaliana than in T. halophila. Thus A. thaliana appears to spend more energy on Na export while nevertheless accumulating more Na than T. halophila. It is proposed that limitation of Na influx is the main mechanism by which T. halophila secures low net Na accumulation in saline conditions. This strategy provides the basis for a positive balance between growth and net Na uptake rates, which is essential for survival in high salt.     

Rapid regulation of Na+ fluxes and ammonia excretion in response to acute environmental hypoxia in the Amazonian oscar, Astronotus ocellatus

The Amazonian oscar is extremely resistant to hypoxia, and tolerance scales with size. Overall, ionoregulatory responses of small ( approximately 15 g) and large oscars ( approximately 200 g) to hypoxia were qualitatively similar, but the latter were more effective. Large oscars exhibited a rapid reduction in unidirectional Na(+) uptake rate at the gills during acute hypoxia (Po(2) approximately 10 mmHg), which intensified with time (7 or 8 h); Na(+) efflux rates were also reduced, so net balance was little affected. The inhibitions were virtually immediate (1st h) and preceded a later 60% reduction (at 3 h) in gill Na(+)-K(+)-ATPase activity, reflected in a 60% reduction in maximum Na(+) uptake capacity without change in affinity (Km) for Na(+). Upon acute restoration of normoxia, recovery of Na(+) uptake was delayed for 1 h. These data suggest that dual mechanisms may be involved (e.g., immediate effects of O(2) availability on transporters, channels, or permeability, slower effects of Na(+)-K(+)-ATPase regulation). Ammonia excretion appeared to be linked indirectly to Na(+) uptake, exhibiting a Michaelis-Menten relationship with external [Na(+)], but the Km was less than for Na(+) uptake. During hypoxia, ammonia excretion fell in a similar manner to Na(+) fluxes, with a delayed recovery upon normoxia restoration, but the relationship with [Na(+)] was blocked. Reductions in ammonia excretion were greater than in urea excretion. Plasma ammonia rose moderately over 3 h hypoxia, suggesting that inhibition of excretion was greater than inhibition of ammonia production. Overall, the oscar maintains excellent homeostasis of ionoregulation and N-balance during severe hypoxia.     

Mechanism of sodium uptake in PNA negative MR cells from rainbow trout, Oncorhynchus mykiss as revealed by silver and copper inhibition

The rate of acid-stimulated and phenamil-sensitive sodium (Na(+)) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA(-) MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na(+) uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H(+)-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na(+) transport in fish). In contrast, Na(+) uptake in PNA(-) MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0 mM external Na(+). Acid-stimulated Na(+) transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na(+) transport in fish, were also responsible for inhibiting acid stimulated Na(+) uptake in PNA(-) MR cells, but by themselves had no effect on basal Na(+) transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na(+) uptake in PNA(-) MR cells in a dose-dependent manner. We also demonstrate rapid (<1 min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA(-) MR cells, but not in PVC. These data lend further support to the idea of a PNA(-) MR cell type as the primary site for Na(+) uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na(+) across the apical surface of the fish gill.