Active calcium ion transport in Xenopuslaevis skin

The skin of intact, free-swimming Xenopus laevis transports Ca²⁺ inwardly in a manner that is proportional to the external [Ca²⁺] up to about 0.3 mmol · l⁻¹, saturates above 0.3 mmol · l⁻¹, and is opposed to the electrochemical gradient. Efflux is relatively constant at external concentrations between 0.016 and 0.6 mmol · l⁻¹; net flux which is negative below 0.125 mmol · l⁻¹ becomes positive above this external [Ca²⁺]. Allometric analysis suggests that both Ca²⁺ influx and efflux scale to the 2/3 power approximately like surface area. There were no significant differences in influx between summer and fall animals; however, efflux was greater in the fall and this resulted in a change from positive balance in the summer to negative balance in the fall. Isolated skins were shown to support a Ca²⁺ uptake rate of nearly 30 nmol · cm⁻² · h⁻¹. The phenylalkylamine verapamil in the apical bathing solution significantly inhibited this at 25 μmol · l⁻¹. The benzothiazepine diltiazem was also effective at 50 μmol · l⁻¹ while the dihydropyradine nifedipine was ineffective up to 100 μmol · l⁻¹. The inorganic ion La³⁺ was effective at blocking Ca²⁺ uptake at 300 μmol · l⁻¹; Ni²⁺ was also effective at 500 μmol · l⁻¹ but Co²⁺ was ineffective up to 500 μmol · l⁻¹. These results suggest that apical calcium channels in Xenopuslaevis skin have properties similar to mammalian L-channels and fish gill Ca²⁺ channels. Accepted: 23 January 1997     

Potential of active excretion of ammonia in three different haline species of crabs

Isolated perfused gills of stenohaline crabs Cancer pagurus adapted to seawater, brackish water-adapted euryhaline shore crabs Carcinus maenas and freshwater-adapted extremely euryhaline Chinese crabs Eriocheir sinensis were tested for their capacity to excrete ammonia. Gills were perfused with haemolymph-like salines and bathed with salines equal in adaptation osmolality. Applying 100 μmol · l⁻¹ NH₄Cl in the perfusion saline and concentrations of NH₄Cl in the bath that were stepwise increased from 0 to 4000 μmol · l⁻¹ allowed us to measure transbranchial fluxes of ammonia along an outwardly as well as various inwardly directed gradients. The gills of all three crab species were capable – to different extents – of active excretion of ammonia against an inwardly directed gradient. Of the three crab species, the gills of Cancer pagurus revealed the highest capacity for active excretion of ammonia, being able to excrete it from the haemolymph (100 μmol · l⁻¹ NH⁺ ₄) through the gill epithelium against ambient concentrations of up to 800 μmol · l⁻¹, i.e. against an eightfold gradient. Carcinus maenas and E. sinensis were able to actively excrete ammonia against approximately fourfold gradients. Within the three crab species, the gills of E. sinensis exhibited the greatest capacity to resist influx at very high external concentrations of up to 4000 μmol · l⁻¹. We consider the observed capacities for excretion of ammonia against the gradient as ecologically meaningful. These benthic crustaceans protect themselves by burying themselves in the sediment, where, in contrast to the water column, concentrations of ammonia have previously been reported that greatly increase haemolymph levels. Electrophysiological results indicate that the permeabilities of the gill epithelia are a clue to understanding the species-specific differences in active excretion of ammonia. During the invasion of brackish water and freshwater, the permeabilities of the body surfaces greatly decreased. The gills of marine Cancer pagurus exibited the greatest permeability (ca. 250 mS cm⁻²), thus representing practically no influx barrier for ions including NH⁺ ₄. We therefore assume that C. pagurus had to develop the strongest mechanism of active excretion of ammonia to counteract influx. On the other hand, freshwater-adapted E. sinensis exhibited the lowest ion permeability (ca. 4 mS cm⁻²) which may reduce passive NH⁺ ₄ influxes at high ambient levels. Accepted: 14 October 1998     

The use of amiloride to uncouple branchial sodium and proton fluxes in the brown trout, Salmo trutta

Resting proton, ammonium and sodium fluxes in Salmo trutta were 492.6 ± 19.5 (n = 29); 122.9 ± 34.2 (n = 28) and 277.1 ± 18.5 (n = 50) μmol · kg⁻¹ · h⁻¹, respectively. The resting transepithelial potential was found to be composed of three successive potentials, the outermost averaging −7.36 ± 0.19mV, the second, −14.3 ± 1.4 mV and the third −37 ± 1.7 mV. Amiloride inhibits the proton, ammonium and sodium fluxes in a dose-dependent manner at concentrations of 0.5 mmol · 1⁻¹ and 0.1 mmol · l⁻¹, but at 0.01 mmol · l⁻¹, proton and ammonium fluxes remained at control levels whilst the sodium was reduced to 70.59 ± 7.29 μmol · kg⁻¹ · h⁻¹. The trans-epithelial potential was effected in a bi-phasic manner by 0.5 mmol · l⁻¹ amiloride. An initial hyperpolarisation of ca. 6 mV was followed by a sustained depolarisation of ca. 14 mV (towards zero) which persisted until the amiloride was washed off the gill. The initial hyperpolarisation was thought to reflect a rapid inhibition of a positive inward sodium current and the subsequent depolarisation was due to the inhibition of a positive outward current (proton) which would abolish the transepithelial potential. However, at 0.01 mmol ·  l⁻¹ only the hyperpolarisation was seen, due to the inhibition of only the inward sodium current. Acetazolamide (0.1 mmol · l⁻¹) was found to have no significant effect on the proton, ammonium and sodium fluxes. These results indicate that the proton and sodium fluxes across the gill of the freshwater trout are not tightly linked. While this suggests that the trout gill resembles the model of Ehrenburg et al. (1985) of sodium uptake in frog skin, the apical potentials measured in the pavement epithelial cell(s) are too low to account for sodium uptake unless the activity of the sodium in the cells is very low. Accepted: 8 August 1996     

The effect of endurance exercise at moderate altitude on serum lipid peroxidation and antioxidative functions in humans

We investigated the effect of training and racing at moderate altitude (MA) on oxidative stress by assessment of serum diene conjugation (DC) and serum antioxidant potential (TRAP). Nine male top level skiers were studied during a national race (20–30 km) at sea level (SL). Thereafter, the athletes trained for 2 weeks at MA, after which they participated in a 20–30 km race at MA. Venous blood samples were taken before and after the race. The DC, indicating early events of lipid peroxi dation, did not change during the race at SL (16 850 vs 15 900 ΔAbsorbance · l⁻¹) or at MA (19 870 vs. 20 630 ΔAbs · l⁻¹). At MA serum DC was higher than at SL both before (25%) and after (30%) the race, the postrace difference being statistically significant (P < 0.05). The TRAP increased during the race at MA (from 1387 to 1943 μmol · 1⁻¹, P  =  0.016), but not at SL (1713 vs 1582 μmol · l⁻¹). These observations would suggest that the level of oxidative stress might be greater during living, training and racing at MA (higher DC levels). Increased TRAP during the race at MA may indicate that the physiological adaptation to extreme acute oxidative stress was altered. The physiological significance of this observation remains to be investigated. Accepted: 18 October 1996     

Water and sodium balances and metabolic physiology of house mice (Mus domesticus) and short-tailed mice (Leggadina lakedownensis) under laboratory conditions

A laboratory study investigated the metabolic physiology, and response to variable periods of water and sodium supply, of two arid-zone rodents, the house mouse (Mus domesticus) and the Lakeland Downs short-tailed mouse (Leggadina lakedownensis) under controlled conditions. Fractional water fluxes for M. domesticus (24 ± 0.8%) were significantly higher than those of L. lakedownensis (17 ± 0.7%) when provided with food ad libitum. In addition, the amount of water produced by M. domesticus and by L. lakedownensis from metabolic processes (1.3 ± 0.4 ml · day⁻¹ and 1.2 ± 0.4 ml · day⁻¹, respectively) was insufficient to provide them with their minimum water requirement (1.4 ± 0.2 ml · day⁻¹ and 2.0 ± 0.3 ml · day⁻¹, respectively). For both species of rodent, evaporative water loss was lowest at 25 °C, but remained significantly higher in M. domesticus (1.1 ± 0.1 mg H₂O · g^−0.122 · h⁻¹) than in L. lakedownensis (0.6 ± 0.1 mg H₂O · g^−0.122 · h⁻¹). When deprived of drinking water, mice of both species initially lost body mass, but regained it within 18 days following an increase in the amount of seed consumed. Both species were capable of drinking water of variable saline concentrations up to 1 mol · l⁻¹, and compensated for the increased sodium in the water by excreting more urine to remove the sodium. Basal metabolic rate was significantly higher in M. domesticus (3.3 ± 0.2 mg O₂ · g^−0.75 · h⁻¹) than in L. lakedownensis (2.5 ± 0.1 mg O₂ · g^−0.75 · h⁻¹). The study provides good evidence that water flux differences between M. domesticus and L. lakedownensis in the field are due to a requirement for more water in M. domesticus to meet their physiological and metabolic demands. Sodium fluxes were lower than those observed in free-ranging mice, whose relatively high sodium fluxes may reflect sodium associated with available food. Accepted: 16 August 1999     

H+ and Cl− secretion in the stomach of the teleost fish, Anguilla anguilla : stimulation by histamine and carbachol

The fundus of an eel stomach was mounted in an Ussing chamber and bathed with control Ringer on the serosal side and with unbuffered solution on the mucosal side. The gastric mucosa exhibited a mucosa negative transepithelial voltage (V _t), a “short circuit” current (I _SC) and a small spontaneous acid secretion rate (J _H). All these parameters were abolished by cimetidine treatment. Bilateral ion substitution experiments in tissues lacking spontaneous acid secretion suggested that a net Cl⁻ transport from serosa to mucosa was responsible for the genesis of the I _SC in the absence of H⁺ secretion. Serosal application of histamine (10⁻⁴ mol · l⁻¹) or carbachol (10⁻⁴ mol · l⁻¹) stimulated both I _SC and J _H. The action of carbachol was independent of histamine. The control as well as the histamine-stimulated I _SC was sensitive to both serosal bumetanide (10⁻⁵ mol · l⁻¹), inhibitor of the Na⁺-K⁺-2Cl⁻ cotransport, and 4,4-diisothiocyano-stilbene-2,2-disulphonic acid (DIDS, 5 · 10⁻⁴ mol · l⁻¹), inhibitor of the Cl⁻-HCO⁻ ₃ exchange, while the I _SC stimulated by carbachol was nullified by serosal DIDS. These data suggested that the non-acidic Cl⁻ uptake across the serosal membrane was linked to the activity of both Na⁺-K⁺-2Cl⁻ cotransport and Cl⁻-HCO⁻ ₃ antiporter; histamine stimulated both transporters while carbachol was limited to the anion exchanger. The finding that the acid secretion was strictly dependent on serosal Cl⁻ and was completely blocked by serosal DIDS suggested that the Cl⁻ accompanying H⁺ secretion entered the cell through the serosal membrane by the Cl⁻-HCO⁻ ₃ exchange. In addition, the acid secretion stimulated by carbachol was also dependent on serosal Na⁺ and sensitive to the application of 5-N-N-dimethyl-amiloride in the serosal bath, suggesting that the increased activity of the Cl⁻-HCO⁻ ₃ during carbachol treatment was linked to the activation of serosal Na⁺-H⁺ exchange. The inhibitory effect of luminal omeprazole (10⁻⁴ mol · l⁻¹) on acid secretion suggested the presence of the H⁺-K⁺ pump on the luminal membrane. Accepted: 18 September 1997     

Insulin and glucagon immunoreactivity during high-intensity exercise under opiate blockade

Eight fit men [maximum oxygen consumption (O_2max) 64.6 (1.9) ml · kg⁻¹ · min⁻¹, aged 28.3 (1.7) years (SE in parentheses) were studied during two treadmill exercise trials to determine the effect of endogenous opioids on insulin and glucagon immunoreactivity during intense exercise (80% O_2max). A double-blind experimental design was used with subjects undertaking the two exercise trials in counterbalanced order. Exercise trials were 20 min in duration and were conducted 7 days apart. One exercise trial was undertaken following administration of naloxone (N; 1.2 mg; 3 ml) and the other after receiving a placebo (P; 0.9% NaCl saline; 3 ml). Prior to each experimental trial a flexible catheter was placed into an antecubital vein and baseline blood samples were collected. Immediately after, each subject received either a N or P bolus injection. Blood samples were also collected after 20 min of continuous exercise (running). Glucagon was higher (P < 0.05), while insulin was lower (P < 0.05), during exercise compared with pre-exercise values in both trials. However, glucagon was higher (P < 0.05) in the P than in the N exercise trial [141.4 (8.3) ng · l⁻¹ vs 127.2 (7.6) ng · l⁻¹]. There were no differences in insulin during exercise between the P and N trials [50.2 (4.3) pmol · l⁻¹ vs 43.8 (5) pmol · l⁻¹]. These data suggest that endogenous opioids may augment the glucagon response during intense exercise. Accepted: 15 June 1996     

Cardio-vascular and ventilatory effects of prostaglandin E2 in the European eel Anguilla anguilla

Prostaglandin E₂ (PGE₂) injection caused an initial decrease in heart rate (HR) (55%), cardiac output (QC) (55%), ventral (VAP, 40%) and dorsal (DAP, 60%) aortic pressures, associated with a marked gill vasoconstriction (100%) and a systemic vasodilation (30%) while cardiac stroke volume (CSV) remained unchanged. It was followed by a progressive recovery of HR, DAP, and vascular resistances to control values while VAP, QC, and CSV significantly increased (20%, 40%, and 40%, respectively). Bradycardia was abolished either by atropine treatment or bilateral section of the cardiac vagus, suggesting that bradycardia could be the consequence of a vagal mechanism triggered by a central action of PGE₂. The increase in QC is due to the increase in CSV, which suggests a positive inotropic action of PGE₂. Infusion with PGE₂ (10⁻⁹ to 10⁻⁵ mol · l⁻¹) did not cause any significant effect on the branchial vasculature in vitro, but when the preparation was pre-treated with adrenaline (10⁻⁸ mol · l⁻⁴), a dose-related vasoconstrictory response to PGE₂ (10⁻⁹ to 10⁻⁵ mol · l⁻¹) occurred. A significant rise (25%) in gill vascular resistance was observed in vivo after treatment with the β-blocker propranolol (2.0 mg · kg⁻¹), suggesting the existence of a vasodilatory β-adrenergic control of the branchial vasculature. PGE₂ caused an apnea followed by a simultaneous tachypnea (50%) and decrease in ventilatory amplitude (40%). The resulting decrease in ventilatory water flow (Q_w) was accompanied by a fall in arterial O₂ tension (Pa O ₂). Under steady Q_w in curarized eels, an initial fall in Pa O ₂ was still observed during the PGE₂-induced gill vasoconstriction. It is concluded that PGE₂ could play a role in the regulation of gas exchanges and blood circulation in particular environmental conditions which require an impairment of gill respiration and a redistribution of blood to the benefit of the systemic vascular bed. Accepted: 3 June 1997     

Active excretion of ammonia across the gills of the shore crab Carcinus maenas and its relation to osmoregulatory ion uptake

The mechanism of transbranchial excretion of total ammonia of brackish-water acclimated shore crabs, Carcinus maenas was examined using isolated, perfused gills. Applying physiological gradients of NH₄Cl (100–200 μmol · l⁻¹) directed from the haemolymph space to the bath showed that the efflux of total ammonia consisted of two components. The saturable component (excretion of NH₄ ⁺) greatly exceeded the linear component (diffusion of NH₃). When an outwardly directed gradient (200 μmol · l⁻¹) was applied, total ammonia in the perfusate was reduced by more than 50% during a single passage of saline through the gill. Effluxes of ammonia along the gradient were sensitive to basolateral dinitrophenol, ouabain, and Cs⁺ and to apical amiloride. Acetazolamide (1 mmol · l⁻¹ basolateral) or Cl⁻-free conditions had no substantial effects on ammonia flux, which was thus independent of both carbonic anhydrase mediated pH regulation and osmoregulatory NaCl uptake. When an inwardly directed gradient (200 μmol · l⁻¹) was employed, influx rates were about 10-fold smaller and unaffected by basolateral ouabain (5 mmol · l⁻¹) or dinitrophenol (0.5 mmol · l⁻¹). Under symmetrical conditions (100 μmol · l⁻¹ NH₄Cl on both sides) ammonia was actively excreted against the gradient of total ammonia, which increased strongly during the experiment and against the gradient of the partial pressure of NH₃. The active excretion rate was reduced to 7% of controls by basolateral dinitrophenol (0.5 mmol · l⁻¹), to 44% by basolateral ouabain (5 mmol · l⁻¹), to 46% by Na⁺-free conditions and to 42% by basolateral Cs⁺ (10 mmol · l⁻¹), indicating basolateral membrane transport of NH₄ ⁺ via the Na⁺/K⁺-ATPase and K⁺-channels and a second active, apically located, Na⁺ independent transport mechanism of NH₄ ⁺. Anterior gills, which are less capable of active ion uptake than posterior gills, exhibited even increased rates of active excretion of ammonia. We conclude that, under physiological conditions, branchial excretion of ammonia is a directed process with a high degree of effectiveness. It even allows active extrusion against an inwardly directed gradient, if necessary. Accepted: 11 March 1998     

Cellular signalling of PCH-induced pigment aggregation in the crustacean Macrobrachium potiuna erythrophores

The cellular system responsible for the transduction of the pigment-concentrating hormone (PCH) signal was investigated in erythrophores of the freshwater shrimp, Macrobrachium potiuna. Dose-response curves to the hormone were determined in the absence and in the presence of several drugs that affect sequential steps of the Ca²⁺-dependent signalling pathway. Additionally, the ability of forskolin to induce pigment dispersion was evaluated. Neomycin sulphate (10⁻⁴ and 10⁻³ mol · l⁻¹), trifluoperazine (10⁻⁵ and 10⁻⁴ mol · l⁻¹), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (10⁻⁷ and 10⁻⁵ mol · l⁻¹) and okadaic acid (10⁻⁷ mol · l⁻¹) significantly (P<0.05) decreased the responses to PCH. However, okadaic acid at low concentration (10⁻⁹ mol · l⁻¹) and cyclosporin A (10⁻⁶ and 10⁻⁵ mol · l⁻¹) did not significantly (P>0.05) affect PCH activity. Forskolin (10⁻⁴ mol · l⁻¹) was able to half-maximally reverse the hormone-induced aggregation. Our results suggest that the pigment-concentrating hormone induces pigment aggregation through a Ca²⁺-dependent pathway with a posteriori phosphatase activation, probably the serine/threonine phosphatase 1. Accepted: 30 June 1997     

Leaf photosynthesis, plant growth and nitrogen allocation in rice under different irradiances

The photosynthetic rates and various components of photosynthesis including ribulose-1,5-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), chlorophyll (Chl), cytochrome (Cyt) f, and coupling factor 1 (CF₁) contents, and sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically under two irradiances, namely, 1000 and 350 μmol quanta · m⁻² · s⁻¹, at three N concentrations. The light-saturated rate of photosynthesis measured at 1800 μmol · m⁻² · s⁻¹ was almost the same for a given leaf N content irrespective of growth irradiance. Similarly, Rubisco content and SPS activity were not different for the same leaf N content between irradiance treatments. In contrast, Chl content was significantly greater in the plants grown at 350 μmol · m⁻² · s⁻¹, whereas Cyt f and CF₁ contents tended to be slightly smaller. However, these changes were not substantial, as shown by the fact that the light-limited rate of photosynthesis measured at 350 μmol · m⁻² · s⁻¹ was the same or only a little higher in the plants grown at 350 μmol · m⁻² · s⁻¹ and that CO₂-saturated photosynthesis did not differ between irradiance treatments. These results indicate that growth-irradiance-dependent changes in N partitioning in a leaf were far from optimal with respect to N-use efficiency of photosynthesis. In spite of the difference in growth irradiance, the relative growth rate of the whole plant did not differ between the treatments because there was an increase in the leaf area ratio in the low-irradiance-grown plants. This increase was associated with the preferential N-investment in leaf blades and the extremely low accumulation of starch and sucrose in leaf blades and sheaths, allowing a more efficient use of the fixed carbon. Thus, morphogenic responses at the whole-plant level may be more important for plants as an adaptation strategy to light environments than a response of N partitioning at the level of a single leaf. Received: 23 February 1997 / Accepted: 8 May 1997     

Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 · 10⁵ cells ml⁻¹, three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l⁻¹) KNO₃ 0.41, Na₂HPO₄ 0.03, MgSO₄ · 7H₂O 0.246, CaCl₂ · 2H₂O 0.11, (in mg l⁻¹) Fe(III)citrate · H₂O 2.62, CoCl₂ · 6H₂O 0.011, CuSO₄ · 5H₂O 0.012, Cr₂O₃ 0.075, MnCl₂ · 4H₂O 0.98, Na₂MoO₄ · 2H₂O 0.12, SeO₂ 0.005 and (in μg l⁻¹]) biotin 25, thiamine 17.5 and B₁₂ 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis. Received: 10 September 1999 / Received revision: 2 December 1999 / Accepted: 3 December 1999     

Role of cyclic nucleotides in pigment translocation within the freshwater shrimp, Macrobrachium potiuna, erythrophore

The participation of cyclic nucleotide-dependent intracellular signalling pathways in the pigment translocation induced by pigment-dispersing hormone (α -PDH) or pigment-concentrating hormone (PCH) was investigated in the erythrophores of the freshwater shrimp, Macrobrachium potiuna. Cholera toxin, forskolin and dibutyryl cyclic adenosine 3′5′ monophosphate (dbcAMP) were able to induce pigment dispersion with effective agonist concentrations for half maximal response (EC₅₀s) of 2.8 · 10⁻¹¹ mol · l⁻¹, 7.0 · 10⁻⁷ mol · l⁻¹ and 3.3 · 10⁻⁷ mol · l⁻¹, respectively. KT5720 (10⁻⁷ mol · l⁻¹ and 10⁻⁶ mol · l⁻¹) significantly shifted the dose response curve to α -PDH to the right. Dibutyryl cyclic guanosine 3′5′ monophosphate (dbcGMP) was ineffective in inducing either pigment aggregation or dispersion. 2′5′ dideoxyadenosine (DDA) and SQ22,536 essentially elicit a pigment-aggregating response in a dose-dependent manner. These effects were not due to the activation of purinergic receptors, since concentrations up to 10⁻⁴ mol · l⁻¹ of adenosine and adenosine triphosphate (ATP), and up to 10⁻³ mol · l⁻¹ of uracil triphosphate (UTP) did not elicit pigment aggregation. In order to verify if PCH decreased cyclic adenosine 3′5′ monophosphate (cAMP) levels, cumulative dose-response curves to PCH in the absence and presence of pertussis toxin and 8-MOM-IBMX were determined. However, neither drug significantly affected PCH activity. The levels of cAMP in the integument cells of M. potiuna were significantly increased (P < 0.05) by α -PDH (10⁻⁷ mol · l⁻¹) and forskolin (10⁻⁶ mol · l⁻¹), but were not affected by PCH (10⁻⁷ or 10⁻¹⁰ mol · l⁻¹). In conclusion, α -PDH seems to elicit pigment dispersion through the activation of a Gs-protein coupled receptor resulting in cAMP increase and cAMP-dependent protein kinase (PKA) activation. Furthermore, although a decrease in cAMP was assumed to be responsible in turn for the action of PCH, such a decrease could not be directly demonstrated. Accepted: 11 August 1998     

Bioenergetics and RNA/DNA ratios in the common carp (Cyprinus carpio ) under hypoxia

Hypoxia caused by eutrophication occurs over large areas in aquatic systems worldwide. Common carp (Cyprinus carpio) exposed to hypoxia (1 mg · O₂ · l⁻¹ and 2 mg · O₂ · l⁻¹) for 1 week showed a significant reduction in feeding rate, respiration rate, faecal production and nitrogenous excretion compared to those maintained at normoxia (7 mg · O₂ · l⁻¹). Fish exposed to hypoxia showed negative scope for growth (SfG), but no significant difference in the specific growth rate was revealed after 1 week in both hypoxic groups. A significant reduction in RNA/DNA ratio was, however, clearly evident in the white muscle of the 1 mg · O₂ · l⁻¹ treatment group, but not in the 2 mg · O₂ · l⁻¹ treatment group. Both specific growth rate and RNA/DNA ratio were significantly reduced when fish were exposed to severe hypoxia (0.5 mg · O₂ · l⁻¹) for 4 weeks. At all levels of hypoxia, growth reduction was accompanied by a significant decrease in RNA/DNA ratio in white muscle. Covariance analysis showed no significant difference between the slope of RNA/DNA ratio and growth rate under normoxic conditions and 0.5 mg · O₂ · l⁻¹ for 4 weeks (F=1.036, P > 0.326), as well as 1.0 mg · O₂ · l⁻¹ and 2.0 mg · O₂ · l⁻¹ for 1 week (F = 0.457, P > 0.5), indicating that the RNA/DNA ratio serves as a biomarker of growth under all oxygen levels, at least under controlled experimental conditions. SfG also appears to be more sensitive than the RNA/DNA ratio in responding to hypoxia in fish. Accepted: 15 September 2000     

Ca2+ regulation of heart contractility in Octopus

Isometric force development of electrically paced preparations isolated from the systemic heart of Octopus vulgaris were utilized to examine the regulation of contractility by Ca²⁺. Increases in extracellular Ca²⁺, to the physiological level, resulted in enhancement of twitch force. For instance, at 36 beats · min⁻¹ an increase in Ca²⁺ from 3 to 9 mmol · l⁻¹ resulted in a threefold increase in twitch force development. When steady-state contraction at 12 beats · min⁻¹ was followed by a rest period of either 5 or 10 min, the first contraction always exhibited either an increase in twitch force or stayed unchanged such that post-rest twitch force was about 133% of the last value in the steady-state train. Ryanodine (12.5 μmol · l⁻¹), which is considered to be a specific inhibitor of the Ca²⁺ storage and release capabilities of the sarcoplasmic reticulum (SR), was applied to further assess Ca²⁺ handling. Twitch force fell to about 22% of the preteatment level in preparations paced at either 12 or 36 beats · min⁻¹. In all preparations the frequency transition from 12 to 36 beats · min⁻¹ was associated with an increase in resting tension. The␣increase␣was 37 ± 14% prior to ryanodine treatment and was significantly elevated to 127 ± 33% following treatment. When steady-state contraction at 36 beats · min⁻¹ was followed by a rest period of 10 s, the first contraction was not significantly different from the last beat in the train prior to ryanodine; however, with ryanodine treatment, post-rest twitch force development significantly decreased. Twitch force development was regular at pacing rates of up to 300 beats · min⁻¹. Twitch force was maintained up to rates of 84 beats · min⁻¹ but␣decreased thereafter and reached a value of about 10% at 300 beats · min⁻¹. Resting tension increased substantially as frequency was elevated from 12 to 36 beats · min⁻¹ and then gradually increased as frequency was further elevated to 180 beats · min⁻¹. In conclusion, the Octopus ventricle is dependent upon extracellular Ca²⁺ for contraction. A post-rest potentiation of force development, the negative impact of ryanodine, and the ability to respond regularly at high pacing rates imply a strong reliance on the SR in Ca²⁺ cycling based on criteria established for vertebrate hearts. Accepted: 19 January 1997     

Protective effects of prostaglandins in the isolated gastric mucosa of the eel, Anguilla anguilla

The protective effect of endogenous prostaglandins on the fish gastric mucosa was evaluated by studying the effect of indomethacin and aspirin, known cyclooxigenase inhibitors, on the mucosal ulceration in the isolated gastric sacs of Anguilla anguilla. Gastric sacs devoid of muscle layers were incubated in the presence of indomethacin (10⁻⁴ mol · l⁻¹) or aspirin (10⁻⁴ mol · l⁻¹) in different experimental conditions. Both the anti-inflammatory drugs produced ulcers, but the effects were more severe in the presence of histamine and in the absence of HCO₃ ⁻ in the incubation bath. The effects of prostaglandin E₂ (PGE₂) on acid secretion rate (J_H) and on alkaline secretion rate (J_OH) were evaluated (with the aid of the pH stat method) in isolated gastric mucosa mounted in Ussing chambers. We found that PGE₂ (10⁻⁸–10⁻⁵ mol · l⁻¹) increased J_H in a dose-dependent manner. In tissues pretreated with luminal omeprazole (10⁻⁴ mol · l⁻¹), PGE₂ stimulated gastric alkaline secretion. It was nullified by serosal removal of HCO₃ ⁻ or Na⁺ and by serosal ouabain (10⁻⁴ mol · l⁻¹). These results suggested that prostaglandins also exert their protective effects in fish gastric mucosa. This protection seems partially due to a stimulation of exogenous HCO₃ ⁻ transport from the serosal to the mucosal side. It is likely that this transport is an active transcellular mechanism coupled to Na⁺ transport. Accepted: 14 April 2000     

Detoxification of environmental sulfide to sulfane sulfur in the intertidal sipunculid Phascolosoma arcuatum

The capability of Phascolosoma arcuatum to detoxify sulfide in anaerobic conditions was examined. Sulfane sulfur, which underwent cold cyanolysis, was the major excretory end product of sulfide detoxification during anoxia. Thiosulfate was not excreted into the external medium. Instead, it was absorbed by P. arcuatum and its absorption was stimulated by the presence of sodium sulfide (Na₂S) in the incubation medium. The effective formation and excretion of sulfane sulfur by P.␣arcuatum required the presence of both Na₂S and sodium thiosulfate (Na₂S₂O₃). Results obtained indicate that rhodanese might be involved in sulfide detoxification in this sipunculid. Rhodanese could act as a catalyst in the transfer of sulfur atoms from thiosulfate to HS⁻. The body wall and the introvert were the main sites of sulfide detoxification. However, it is unlikely that epibiotic bacteria associated with the outside surface of the worm were involved in the detoxification process. A time-course study on the contents of thiosulfate and sulfane sulfur in the body wall of P. arcuatum incubated anaerobically in the presence of Na₂S + Na₂S₂O₃ verified that thiosulfate absorbed was utilized to detoxify sulfide to sulfane sulfur. Accepted: 24 October 1996     

Uptake of 3-O-methyl-D-[U-14C]glucose into brain of rainbow trout: possible effects of melatonin

The influx of glucose into the brain and plasma glucose disappearance were estimated in rainbow trout (Oncorhynchus mykiss) intravenously injected (1 ml · kg⁻¹ body weight) with a single dose (15 μCi · kg⁻¹ body weight) of 3-O-methyl-D-[U-¹⁴C]glucose ([U-¹⁴C]-3-OMG) at different times (2–160 min), and after intravenous injection at 15 min of increased doses (10–60 μCi · kg⁻¹ body weight) of [U-¹⁴C]-3-OMG. Brain and plasma radiotracer concentrations were measured, and several kinetic parameters were calculated. The apparent brain glucose influx showed a maximum after 15–20 min of injection then decreased to a plateau after 80 min. Brain distribution space of 3-OMG increased from 2 min to 20 min reaching equilibrium from that time onwards at a value of 0.14 ml · g⁻¹. The unidirectional clearance of glucose from blood to brain (k₁) and the fractional clearance of glucose from brain to blood (k₂) were estimated to be 0.093 ml · min⁻¹ · g⁻¹, and 0.867 min⁻¹, respectively. A linear increase was observed in brain and plasma radiotracer concentrations when increased doses of [U-¹⁴C]-3-OMG were used. All these findings support a facilitative transport of glucose through the blood-brain barrier of rainbow trout with characteristics similar to those observed in mammals. The injection of different doses of melatonin (0.25–1.0 mg · kg⁻¹) significantly increased brain glucose influx suggesting a possible role for melatonin in the regulation of glucose transport into the brain. Accepted: 26 January 2000     

The effect of elevated CO2 concentrations on K+ and anion channels of Vicia faba L. guard cells

The effects of elevated CO₂ concentrations on stomatal movement, anion- and K⁺-channel activities were examined in guard cells from epidermal strips of Vicia faba. Membrane voltage was measured using intracellular, double-barrelled microelectrodes and ion-channel currents were recorded under voltage clamp during exposure to media equilibrated with ambient (350 μl · l⁻¹), 1000 μl · l⁻¹ and 10 000 μl · l⁻¹ CO₂ in 20% O₂ and 80% N₂. The addition of 1000 μl · l⁻¹ CO₂ to the bathing solution caused stomata to close with a halftime of approx. 40 min, and with 10 000 μl · l⁻¹ CO₂ closure occurred with a similar time course. Under voltage clamp, exposure to 1000 μl · l⁻¹ and 10 000 μl · l⁻¹ CO₂ resulted in a rapid increase (mean, 1.5 ± 0.2-fold, n = 8; range 1.3- to 2.5-fold) in the magnitude of current carried by outward-rectifying K⁺ channels (I_K,out). The effect of CO₂ on I_K,out was essentially complete within 30 s and was independent of clamp voltage, but was associated with 25–40% (mean, 30 ± 4%) decrease in the halftime for current activation. Exposure to CO₂ also resulted in a four-fold increase in background current near the free-running membrane voltage, recorded as the instantaneous current at the start of depolarising and hyperpolarising voltage steps, and a decrease in the magnitude of current carried by inward-rectifying K⁺ channels (I_K,in). The effect of CO₂ on I_K,in was generally slower than on I_K,out; it was allied with a transient acceleration of its activation kinetics during the first 60–120 s of treatment; and it was associated with a negative shift in the voltage-sensitivity of gating over a period of 3–5 min. Measurements carried out to isolate the background currents attributable to anion channels (I_Cl), using tetraethylammonium chloride and CsCl, showed that CO₂ also stimulated I_Cl and dramatically altered its relaxation kinetics. Within the timeframe of CO₂ action at the membrane, no significant effect was observed on cytosolic pH, measured using the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyflourescein (BCECF) and ratio fluorescence microphotometry. These results are broadly consistent with the pattern of guard-cell response to abscisic acid, and indicate that guard cells control both anion and K⁺ channels to achieve net solute loss in CO₂. By contrast with the effects of abscisic acid, however, the data indicate that CO₂ action is not mediated through changes in cytosolic pH and thereby implicate new and, as yet, unidentified pathway(s) for channel regulation in the guard cells. Received: 8 January 1997 / Accepted: 28 February 1997     

The relationship between plasma potassium concentration and muscle torque during recovery following intense exercise

The present study investigated the relationship between plasma potassium ion concentration ([K⁺]) and skeletal muscle torque during three different 15-min recovery periods after fatigue induced by four 30-s sprints. Four males and one female completed the multiple sprint exercise on three separate days; recovery was passive, i.e. no cycling exercise (PRec), active cycling at 30% peak oxygen consumption O_2peak (30% Rec) and active cycling at 60% O_2peak (60% Rec). Plasma [K⁺] was measured from blood sampled from an antecubital vein of subjects at rest and at 0, 3, 5, 10 and 15 min into each recovery. Isokinetic leg strength was measured at rest and at 1, 6, 11 and 16 min during each recovery. Following the exhaustive sprints, [K⁺] increased significantly from an average mean (SEM) resting value of 3.81 (0.07) mmol · l⁻¹ to 4.48 (0.19) mmol · l⁻¹ (P < 0.01). In all recovery conditions, plasma [K⁺] returned to resting levels within 3 min following the fourth sprint. However, in the two active recovery conditions plasma [K⁺] increased over the remainder of the recovery periods to 4.36 (0.12) mmol · l⁻¹ in the 30% Rec condition and 4.62 (0.12) mmol · l⁻¹ in the 60% Rec condition, the latter being significantly higher than the former (P < 0.01). The maximum torque measured following the sprints decreased significantly, on average, to 61.1 (8.36)% of peak levels (P < 0.01). After 15 min of recovery, maximum torque was highest in the 30% Rec condition at 92.13 (3.06)% of peak levels (P < 0.01), compared to 85.23 (3.64)% and 85.71 (0.82)% for the PRec and 60% Rec conditions, respectively. In contrast to the significant differences in plasma [K⁺] across all three recovery conditions, muscle torque recovery was significantly different in only the 30% Rec condition. In summary, recovery of peak levels of muscle torque following fatiguing exercise does not appear to follow changes in plasma [K⁺]. Accepted: 18 October 1996