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     

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     

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     

Signal transduction in isolated fat body from the cockroach Blaptica dubia exposed to hypertrehalosaemic neuropeptide

Hypertrehalosaemic hormones stimulate trehalogenesis while inhibiting glycolysis in cockroach fat body. Signal transduction of the hypertrehalosaemic peptide Bld HrTH was examined in isolated fat body of the Argentine cockroach Blaptica dubia with respect to its effects on the increase in trehalose production and decrease in the content of the glycolytic activator fructose 2,6-bisphosphate in the tissue. Cyclic AMP does not seem to be involved in these processes as the cAMP analogue cpt-cAMP and the phosphodiesterase inhibitor IBMX, which both permeate cell membranes, had no effect on either parameter. Octopamine at physiological concentrations (10⁻⁷ mol · l⁻¹) was also ineffective, but at 10⁻⁵ mol · l⁻¹ or above, octopamine stimulated trehalose production although the content of fructose 2,6-bisphosphate in fat body was not affected. Both calcium entry and the release of Ca²⁺ from intracellular stores seem to be involved in the action of the hormone. If Ca²⁺ was omitted from the incubation medium, the hormone stimulated trehalose production less, though still significantly, whereas the hormone effect on fructose 2,6-bisphosphate was completely abolished in the absence of extracellular Ca²⁺. With Ca²⁺ present in the medium, the effect of the hormone on fructose 2,6-bisphosphate could be fully mimicked by the calcium ionophore A23187, suggesting that calcium entry is a␣decisive step in this signalling pathway. Trehalose production, on the other hand, was increased by thimerosal and thapsigargin which increase cytosolic Ca²⁺ from intracellular stores, whereas thimerosal in the absence of extracellular Ca²⁺ increased rather than decreased the content of fructose 2,6-bisphosphate, thus dissociating the two effects, which are normally coordinated by the hormone. Trehalose production and the content of fructose 2,6-bisphosphate were not significantly affected by mepacrine and mellitin, which are known to inhibit, respectively stimulate, phospholipase A₂. Our data suggest that the effects of Bld HrTH on the stimulation of trehalose production and reduction of fructose 2,6-bisphosphate content in fat body are mediated by Ca²⁺, but that different signalling pathways are involved, suggesting that the two processes, although they are functionally linked, could be regulated separately. Accepted: 10 November 1997     

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     

Circadian rhythms in light-evoked responses of the fly's compound eye, and the effects of neuromodulators 5-HT and the peptide PDF

Two sets of wide-field neurons extend neurites into the fly's optic lamina, where monopolar cells receive photoreceptor input. They exhibit immunoreactivity to antibodies raised against either 5-hydroxytryptamine or the crustacean peptide PDH, respectively. Both are proposed whole-field neuromodulators of vision, apparently regulating a circadian rhythm of monopolar cell size. Seeking functional correlates, we have re-examined the electroretinogram for circadian rhythmicity, and for responses to locally injected 5-hydroxytryptamine and peptide. Long-term electroretinogram recordings from Calliphora entrained to a light/dark cycle and then transferred to constant darkness, uncovered a gradual, modest increase during the subjective night in the electroretinogram's ON- and OFF-transients, from the lamina's monopolar cells. Five to twenty nl of 5-hydroxytryptamine (10⁻³ mol · 1⁻¹) injected into the head haemolymph strongly enhanced the electroretinogram transients, an action reversed by 5-hydroxytryptamine antagonists. Injected into the eye, 5-hydroxytryptamine (10⁻⁴ mol · 1⁻¹) had the opposite effect; the rapid onset there suggests direct action, whilst the opposing effect from haemolymph injection suggests a different receptor site. Pigment-dispersing hormone (2.2 × 10⁻⁵ mol · 1⁻¹) injected into the haemolymph increased the electroretinogram transients along a biphasic course, with a slow partial recovery; injected into the eye, it lacked effect. Accepted: 30 May 1999     

Evidence for NO-dependent vasodilation in the trout (Oncorhynchus mykiss ) coronary system

The effects of l-arginine, and its analogues N ^ω-nitro-l-arginine methyl ester and N ^ω-nitro-l-arginine on vascular resistance were investigated in the intact coronary system of an isolated non-working trout heart preparation. l-Arginine, at 10^–8 mol · l^–1induced a slight vasodilatory effect (max 10%). N ^ω-nitro-l-arginine methyl ester and N ^ω-Nitro-l-arginine in the range 10^–8–10^–4 mol · l^–1 caused dose-dependent increases in coronary resistance. The vasodilatory action of l-arginine was abolished when the preparation was pretreated with 10^–4 mol · l^–1 N ^ω-nitro-l-arginine or N ^ω-nitro-l-arginine methyl ester. Nitroprusside alone at 1 mmol · l^–1 induced a maximum vasodilation (30%) of the coronary system. Methylene blue a known inhibitor of guanylate cyclase, induced a strong vasoconstriction (already significant at 10^–5 mol · l^–1) and was able to overcome the vasodilative effect of nitroprusside. The endothelial nitric oxide agonists acetylcholine and serotonin, established in mammalian vessels, also mediate vasodilation in trout coronary system. In 50% of preparations, acetylcholine induced a biphasic response with vasodilation at low concentration (max 15% at 10^–8 mol · l^–1). Serotonin displayed a dose-response vasodilation in the range 10^–8–10^–4 mol · l^–1 (max 20%). These vasodilative effects were reduced or abolished by 10^–4 mol · l^–1 l-NA. These data support the existence of NO-mediated vasodilation mechanisms in the trout coronary system. Accepted: 1 July 1996     

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     

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     

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     

Oxygen consumption and sulfide detoxification in the lugworm Arenicola marina (L.) at different ambient oxygen partial pressures and sulfide concentrations

The lugworm Arenicola marina is a typical inhabitant of intertidal flats. In its L-shaped burrow the animal is exposed to varying concentrations of O₂ and toxic sulfide depending on the tides. The lugworm is able to detoxify sulfide through its oxidation to thiosulfate. When exposed to declining O₂ tensions Arenicola marina reacted as an oxyconformer. In the presence of 25 μmol · l⁻¹ sulfide the respiration was not affected. In contrast, the lugworm consumed significantly less O₂ at any Po₂ in the presence of 200 μmol · l⁻¹ sulfide. Without sulfide anaerobic metabolism started at a Po₂ of approximatedly 10 kPa. Even at high O₂ tensions animals exposed to sulfide produced significantly more anaerobic metabolites compared with the controls. Accordingly the critical value Pc_M, the ambient Po₂ below which anaerobic metabolism starts, was shifted towards normoxia. Since O₂ supply was sufficient for aerobic metabolism, anaerobiosis was induced by sulfide. An influx of sulfide was observed at 25 as well as at 200 μmol · l⁻¹ sulfide. The main product of sulfide detoxification in the lugworm was thiosulfate. Its synthesis increased with ambient Po₂ and depended on the sulfide concentration. Sulfide and thiosulfate were detected in the coelomic fluid, the blood, and the body wall of Arenicola marina. Only about 2% of the ambient O₂ was used for sulfide detoxification at 25 μmol · l⁻¹ sulfide and about 50% at 200 μmol · l⁻¹ sulfide, respectively. Even at the low sulfide concentration Arenicola marina's capacity to detoxify sulfide was too low to maintain a complete aerobic metabolism. Accepted: 19 February 1997     

A phospholipase inhibitor, manoalide, and a G-protein activator, Mas-7, both affect the turnover of phototransductive membranes by crab retinas in darkness With a model for the regulation of rhabdomeral membrane turnover

(1) In vitro retinas of a crab, Leptograpsus, were treated with a phospholipase inhibitor, manoalide, or a G-protein activator, Mas-7. Both drugs address early stages of the phototransduction cascade. (2) Manoalide inhibited the light-dependent reduction of rhabdoms during the `day' phase of the light cycle, but did not induce rhabdom overgrowth. Following a period of darkness manoalide failed to affect the diminution of illuminated rhabdoms. (3) The diminution of rhabdoms that follows photoreceptor depolarisation induced by 100 mmol · l<sup>−1</sup> K<sup>+</sup> in darkness was not affected by 2␣μmol · l<sup>−1</sup> manoalide. (4) When retinas in the `night' phase were treated with Mas-7 in darkness, rhabdom diameters were augmented, concurrently with endocytosis of photoreceptor plasma membranes. (5) The results of combining manoalide and Mas-7 with actinomycin D, U-57908 or okadaic acid, drugs used in previous studies to manipulate steps notionally lower in the transduction cascade, lead to a hypothetical model for the regulation of phototransductive membrane turnover by arthropods. Accepted: 3 October 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     

In vitro studies on calcium absorption from the gastrointestinal tract in␣small ruminants

Unidirectional flux rates of Ca²⁺ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca²⁺ flux rates (J _ms) exceeded respective flux rates in the opposite direction (J _sm) in both species and in all segments of the intestinal tract. This resulted in net Ca²⁺ flux rates␣(J _net = J _ms − J _sm) ranging between −2 and 9 nmol · cm⁻² · h⁻¹ in sheep and between 10 and 15 nmol cm⁻² · h⁻¹ in goats. In sheep, only J _net in jejunum, and in goats, J _netin duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant J _net of Ca²⁺ of around 5 nmol · cm⁻² · h⁻¹ could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca²⁺ absorption clearly indicates the presence of active mechanisms for Ca²⁺ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca²⁺ absorption in goat rumen. J _net of Ca²⁺ across goat rumen epithelia was significantly reduced by 1 mmol · l ⁻¹ verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in small ruminants. Stimulation of active Ca²⁺ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca²⁺ transport as described for the upper small intestines of monogastric species. Accepted: 31 July 1996     

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     

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     

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     

Effect of manipulation of the renin-angiotensin system in control of drinking in juvenile Atlantic salmon (Salmosalar L) in fresh water and after transfer to sea water

Drinking in Atlantic salmon (Salmo salar) juveniles was investigated in fresh water and following transfer to sea water. There was a significant effect of fish size on drinking, and smolts (20–30 g) imbibed about ten times less water than alevins of 0.2–0.3 g. Freshwater smolts drank at a rate of 0.15 ± 0.03 ml · kg⁻¹ · h⁻¹ and administration of doses of 10 or 20 mg · kg⁻¹ of papaverine (stimulator of the renin- angiotensin system RAS) or [Asn¹, Val⁵]-Angiotensin II (0.4 μmol · kg⁻¹) resulted in significant increases in drinking, while administration of the angiotensin converting enzyme inhibitor, enalapril (50 mg · kg⁻¹) had no effect on drinking. Transfer of Atlantic salmon smolts to 1/3, 2/3 and full strength sea water resulted in significant increases in drinking to 1.06 ± 0.12, 1.24 ± 0.0.16 and 3.89 ± 0.28 ml · kg⁻¹ · h⁻¹, respectively. In sea water, stimulation of the endogenous RAS by administration of papaverine (20 mg · kg⁻¹) resulted in a 20% increase in drinking, while administration of enalapril to doses of 50 and 200 mg · kg⁻¹ lowered drinking to 1.99 ± 0.48 and 0.32 ± 0.06 ml · kg⁻¹ · h⁻¹, respectively. All treatments were without effect on blood plasma levels of Na⁺ and Cl⁻ in fresh water, while in sea water smolts both stimulation and inhibition of drinking resulted in hemoconcentration of Na⁺ and Cl⁻. The role of the renin angiotensin system in control of drinking and hydromineral balance in Atlantic salmon is discussed. Accepted: 27 February 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     

Catecholamine release in heat-stressed Antarctic fish causes proton extrusion by the red cells

Two species of Antarctic fish were stressed by moving them from seawater at −1 °C to seawater at 10 °C and holding them for a period of 10 min. The active cryopelagic species Pagothenia borchgrevinki maintained heart rate while in the benthic species Trematomus bernacchii there was an increase in heart rate. Blood pressure did not change in either species. Both species released catecholamines into the circulation as a consequence of the stress. P. borchgrevinki released the greater amounts, having mean plasma concentrations of 177 ± 54 nmol · l⁻¹ noradrenaline and 263 ± 131 nmol · l⁻¹ adrenaline at 10 min. Plasma noradrenaline concentrations rose to 47 ± 14 nmol · l⁻¹ and adrenaline to 73 ± 28 nmol · l⁻¹ in T. bernacchii. Blood from P. borchgrevinki was tonometered in the presence of isoprenaline. A fall in extracellular pH suggests the presence of a Na⁺/H⁺ antiporter on the red cell membrane, the first demonstration of this in an Antarctic fish. Treatment with the β-adrenergic antagonist drug sotalol inhibited swelling of red blood cells taken from temperature-stressed P. borchgrevinki, suggesting that the antiporter responds to endogenous catecholamines. Accepted: 22 January 1998