Haemolymph oxygen transport and acid-base status in Glyptonotus antarcticus Eights

Haemolymph samples were withdrawn from routinely active male intermoult Glyptonotus held at 0 ± 0.5°C, and analysed for blood-gas and acid-base variables. In both the arterialised (a) and venous (v) haemolymph, over 50% of the oxygen was transported as dissolved oxygen at PaO₂ and PvO₂ levels of 12.0 ± 1.15 and 7.70 ± 1.89 kPa, respectively. The maximum oxygen-carrying capacity of the haemocyanin (C^maxHcO₂) was relatively low at 0.19 ± 0.05 mmol l⁻¹, accompanied by relatively low protein and [Cu²⁺] levels indicating low circulating haemocyanin concentrations. Arterialised haemolymph had a mean pH of 7.88 ± 0.02(6) at a PCO₂ of 0.12 ± 0.01(6) kPa and a bicarbonate level of 12.95 ± 0.80(6) mequiv l⁻¹ with small differences in PCO₂ and pH between arterial and venous haemolymph. The non-bicarbonate buffering capacity of Glyptonotus haemolymph was low at −2.0 mequiv l⁻¹ HCO₃ ⁻ pH unit⁻¹. Haemolymph [l-lactate] and [d-glucose] levels were similar at < 1 mmol l⁻¹ in animals held in the laboratory and those sampled in Antarctica. The blood-gas and acid-base status of Glyptonotus haemolymph may be a reflection of the low and stable temperatures experienced by this Antarctic crustacean. Received: 14 August 1996 / Accepted: 3 November 1996     

Copper exposure impairs intra- and extracellular acid-base regulation during hypercapnia in the fresh water rainbow trout (Oncorhynchus mykiss)

In order to evaluate the impact of water-borne copper on acid-base regulation in fresh water rainbow trout, chronically cannulated fish were exposed to copper (0.6 mg 1⁻¹), hypercapnia (water PCO₂ of 6 mmHg) or a combination of copper and hypercapnia, while a fourth untreated group served as the control. Blood samples obtained at 0 h, 4 h and 24 h were analysed for acid-base status, ion concentrations and respiratory parameters. Tissue samples from caudal skeletal muscle, liver and gill filaments were examined for intracellular acid-base status, ion- and water contents, and copper concentration. Exposure to copper alone elicited a small extracellular metabolic alkalosis, no changes in arterial PO₂, and a minor decrease in plasma ion concentrations. Hypercapnia alone increased arterial PCO₂ from approximately 2 mmHg to 7.2 mmHg, but the extracellular respiratory acidosis present at 4 h was almost completely compensated at 24 h due to an increase in plasma bicarbonate concentration [HCO₃ ⁻] from 8.1 mM to 24.4 mM. Combined exposure to hypercapnia and copper resulted in a slightly larger acidosis at 4 h, and the fish failed to restore extracellular pH at 24 h, because plasma [HCO₃ ⁻] only increased to 16.3 mM. Fish exposed to hypercapnia and copper also showed a delayed recovery of intracellular pH in skeletal muscle, compared to fish exposure to hypercapnia only. Thus, copper exposure impaired both extracellular and intracellular acid-base regulation during hypercapnia. When seen in connection with only minor effects of copper on osmoregulatory and respiratory parameters, the reduced ability to regulate acid-base suggests that acid-base regulation may be one of the most copper-sensitive branchial functions. Accepted: 18 August 1998     

Acid–base regulation in the plainfin midshipman (Porichthys notatus): an aglomerular marine teleost

The plainfin midshipman (Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid–base regulation during respiratory acidosis either by facilitating or constraining HCO₃ ⁻ accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid–base status during exposure to increasing levels of hypercapnia (nominally 1–5% CO₂). Fish exhibited a well-developed ability to increase plasma HCO₃ ⁻ levels with values of 39.8 ± 2.8 mmol l⁻¹ being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO₂ = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO₃ ⁻ during hypercapnia was associated with marked increases in branchial net acid excretion (J _NETH⁺) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in J _NETH⁺ was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na⁺/K⁺-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO₃ ⁻ concentrations of 92.2 ± 4.8 mmol l⁻¹. At the highest level of hypercapnia, rectal fluid HCO₃ ⁻ levels were increased significantly to 141.8 ± 7.4 mmol l⁻¹ but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l⁻¹). Thus, the impressive accumulation of plasma HCO₃ ⁻ to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO₃ ⁻ excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO₃ ⁻ concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO₃ ⁻. The apparent inability of P. notatus to effectively excrete HCO₃ ⁻ following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts.     

Interprotein metal ion exchange between cadmium-carbonic anhydrase and apo- or zinc-metallothionein

 − 1  s^− 1 at 25 °C and pH 7.4 in Tris.HCl buffer and 0.1 M KCl. At 25 °C, Zn₇-metallothionein also exchanged metal ions with Cd-carbonic anhydrase with a rate constant of 0.33 ± 0.02 M^− 1 s^− 1 to reconstitute enzymatically active protein. Cd-carbonic anhydrase reacted within the time of mixing with the peptide sequence 49–61 of rabbit metallothionein 2 which contains four cysteinyl residues, leading to the exchange of most of the Cd²⁺ into the peptide. At pH 7.4 and 25 °C, Cd²⁺ has higher affinity for apometallothionein than for the apo-peptide. Received: 25 February 1999 / Accepted: 17 September 1999     

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     

The role of external carbonic anhydrase in the photosynthetic use of inorganic carbon in the deep-water alga Phyllariopsis purpurascens (Laminariales, Phaeophyta)

Mechanisms of inorganic carbon assimilation were investigated in the deep-water alga Phyllariopsis purpurascens (C. Agardh) Henry et South (Laminariales, Phaeophyta). The gross photosynthetic rate as a function of external pH, at a constant concentration of 2 mM dissolved inorganic carbon (DIC), decreased sharply from pH 7.0 to 9.0, and was not substantially different from 0 above pH 9.0. These data indicate that P. purpurascens is inefficient in the use of external HCO₃ ⁻ as a carbon source in photosynthesis. Moreover, the photosynthetic rate as a function of external DIC and the highest pH (9.01 ± 0.07) that this species can achieve in a closed system were consistent with a low capacity to use HCO₃ ⁻, in comparison to many other species of seaweeds. The role of external carbonic anhydrase (CA; EC 4.2.1.1) on carbon uptake was investigated by measuring both the HCO₃ ⁻-dependent O₂ evolution and the CO₂ uptake, at pH 5.5 and 8.0, and the rate of pH change in the external medium, in the presence of selected inhibitors of extra- and intracellular CA. Photosynthetic DIC-dependent O₂ evolution was higher at pH 5.5 (where CO₂ is the predominant form of DIC) than at pH 8.0 (where the predominant chemical species is HCO₃ ⁻). Both intra- and extracellular CA activity was detected. Dextran-bound sulfonamide (DBS; a specific inhibitor of extracellular CA) reduced the photosynthetic O₂ evolution and CO₂ uptake at pH 8.0, but there was no effect at pH 5.5. The pH-change rate of the medium, under saturating irradiance, was reduced by DBS. Phyllariopsis purpurascens has a low efficiency in the use of HCO₃ ⁻ as carbon source in photosynthesis; nevertheless, the ion can be used after dehydration, in the external medium, catalyzed by extracellular CA. This mechanism could explain why the photosynthetic rate in situ was higher than that supported solely by the diffusion of CO₂ from seawater. Received: 6 March 1998 / Accepted: 22 June 1998     

Water influx and efflux in free-flying pigeons

We used tritium-labeled water to measure total body water, water influx (which approximated oxidative water production) and water efflux in free-flying tippler pigeons (Columba livia) during flights that lasted on average 4.2 h. At experimental air temperatures ranging from 18 to 27 °C, mean water efflux by evaporation and excretion [6.3 ± 1.3 (SD) ml · h⁻¹, n = 14] exceeded water influx from oxidative water and inspired air (1.4 ± 0.7 ml · h⁻¹, n = 14), and the birds dehydrated at 4.9 ± 0.9 ml · h⁻¹. This was not significantly different from gravimetrically measured mass loss of 6.2 ± 2.1 g · h⁻¹ (t = 1.902, n = 14, P>0.05). This flight-induced dehydration resulted in an increase in plasma osmolality of 4.3 ± 3.0 mosmol · kg⁻¹ · h⁻¹ during flights of 3–4 h. At 27 °C, the increase in plasma osmolality above pre-flight levels (ΔP _osm = 7.6±4.29 mosmol · kg⁻¹ · h⁻¹, n = 6) was significantly higher than that at 18 °C (ΔP _osm = 0.83±2.23 mosmol · kg⁻¹ · h⁻¹, (t = 3.43, n = 6, P < 0.05). Post-flight haematocrit values were on average 1.1% lower than pre-flight levels, suggesting plasma expansion. Water efflux values during free flight were within 9% of those in the one published field study (Gessaman et al. 1991), and within the range of values for net water loss determined from mass balance during wind tunnel experiments (Biesel and Nachtigall 1987). Our net water loss rates were substantially higher than those estimated by a simulation model (Carmi et al. 1992) suggesting some re-evaluation of the model assumptions is required. Accepted: 8 April 1997     

Effects of NH4 +, NO3 − and HCO3 − on apoplast pH in the outer cortex of root zones of maize, as measured by the fluorescence ratio of fluorescein boronic acid

A fluorimetric ratio technique was elaborated to measure apoplastic pH in the outer root cortex of maize (Zea mays L.) grown hydroponically. A newly synthesized fluorescent probe, fluorescein boronic acid (pK_a = 5.48), which covalently binds to the cell wall of the outer cell layers, was used. Under conditions of saturating ion concentrations the apoplastic pH was determined along the root axis ranging from 1 to 30 mm behind the root tip. Apoplastic pH was recorded for root segment areas (1 mm²), and pH values of high statistical significance were obtained. With an external solution of pH 5, the apoplastic pH was about pH 5.1 in the division zone, between pH 4.8 and 4.9 in the elongation region and about pH 4.9 in the root hair zone. At an external pH of 8.6, the difference between the external pH and the apoplastic pH was considerably more, with a pH of 5.2–5.3 in all root zones. Addition of 1 mM NH₄ ⁺ caused a small apoplastic pH decrease (0.05 of a pH unit) in all root zones. Apoplastic alkalization upon application of 6 mM NO₃ ⁻ was highest (0.3 of a pH unit) in the zone where root hairs emerge; in the division and early elongation zones, apoplastic pH increased only transiently. In the presence of 10 mM HCO₃ ⁻, NO₃ ⁻ elicited a higher and persistent alkalization (0.06–0.25 of a pH unit) in all root zones. Application of fusicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, while inhibition of the H⁺-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the root axis upon differential N supply and application of HCO₃ ⁻ provide evidence that this new pH technique is a useful tool with which to measure apoplastic pH, and in future may permit measurements at microsites at the cell level by use of microscope imaging. Received: 26 August 1998 / Accepted: 4 May 1999     

Gas exchange and heart rate in the harbour porpoise, Phocoena phocoena

The respiratory physiology, heart rates and metabolic rates of two captive juvenile male harbour porpoises (both 28 kg) were measured using a rapid-response respiratory gas analysis system in the laboratory. Breath-hold durations in the laboratory (12 ± 0.3 s, mean ± SEM) were shorter than field observations, although a few breath-holds of over 40 s were recorded. The mean percentage time spent submerged was 89 ± 0.4%. Relative to similarly-sized terrestrial mammals, the respiratory frequency was low (4.9 ± 0.19 breaths · min⁻¹) but with high tidal volumes (1.1 ± 0.01 l), enabling a comparatively high minute rate of gas exchange. Oxygen consumption under these experimental conditions (247 ± 13.8 ml O₂ · min⁻¹) was 1.9-fold higher than predicted by standard scaling relations. These data together with an estimate of the total oxygen stores predicted an aerobic dive limit of 5.4 min. The peak end-tidal O₂ values were related to the length of the previous breath-hold, demonstrating the increased oxygen uptake from the lung for the longer dives. Blood oxygen capacity was 23.5 ± 1.0 ml · 100 ml⁻¹, and the oxygen affinity was high, enabling rapid oxygen loading during ventilation. Accepted: 11 August 1999     

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     

Anion uptake and acid-base and ionic effects during isolated and combined exposure to hypercapnia and nitrite in the freshwater crayfish, Astacus astacus

Nitrite influx into crayfish showed saturation kinetics, supporting a carrier-mediated uptake. Addition of 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS: at 10⁻⁵, 10⁻⁴ and 10⁻³M) and bumetanide (at 10⁻⁵ M and 10⁻⁴ M) to the ambient water did not significantly affect nitrite influx. Rather than suggesting that neither Cl⁻/HCO₃ ⁻ exchange nor K⁺/Na⁺/2Cl⁻ cotransport were involved in the transport, this may reflect that the gill cuticle has a low permeability to the pharmacological agents, or that the sensitivity of the transport mechanism to the inhibitors is low. Nitrite accumulation in the haemolymph was significantly decreased during hypercapnic conditions compared to normocapnic conditions. This supports the idea that an acid–base regulatory decrease in Cl⁻(influx)/HCO₃ ⁻ (efflux) induced by hypercapnia should decrease NO₂ ⁻ uptake if NO₂ ⁻ and Cl⁻ share this uptake route. The respiratory acidosis caused by exposure to hypercapnia alone was partially compensated by HCO₃ ⁻ accumulation in the haemolymph. Combined exposure to hypercapnia and nitrite improved pH recovery, mainly by augmenting the [HCO₃ ⁻] increase, but also by decreasing haemolymph PCO₂. Exposure to nitrite in normocapnic water induced an initial increase in haemolymph [HCO₃ ⁻] and later also a decrease in PCO₂. Thus, the improved acid-base compensation during combined hypercapnia and nitrite exposure was an amplification of this nitriteinduced response. Haemolymph base excess rose much more than haemolymph [Ca], suggesting that transfer of acid-base equivalents between animal and water was more important than H⁺ buffering by exoskeletal CaCO₃ in mediating the increase in haemolymph [HCO₃ ⁻]. Accepted: 27 June 2000     

Dopamine, sensory discharge, and stimulus interaction with CO2 and O2 in cat carotid body

It is hypothesized that carotid bodychemosensory activity is coupled to neurosecretion. The purpose of thisstudy was to examine whether there was a correspondence between carotidbody tissue dopamine (DA) levels and neuronal discharge (ND) measured from the carotid sinus nerve of perfused cat carotid bodies and tocharacterize interaction betweenCO₂ andO₂ in these responses. ND andtissue DA were measured after changing from normoxic, normocapnic control bicarbonate buffer (PO₂>120 Torr, PCO₂ 25-30 Torr, pH ~ 7.4) to normoxic hypercapnia(PCO₂ 55-57 Torr, pH7.1-7.2) or to hypoxic solutions(PO₂ 30-35 Torr) withnormocapnia (PCO₂ 25-30 Torr, pH ~ 7.4) or hypocapnia (PCO₂10-15 Torr, pH 7.6-7.8). Similar temporal changes for ND and tissue DA were found for all of the stimuli, although there was a much different proportional relationship fornormoxic hypercapnia. Both ND and DA increased above baseline valuesduring flow interruption and normocapnic hypoxia, and both decreasedbelow baseline values during hypoxic hypocapnia. In contrast, normoxichypercapnia caused an initial increase in ND, from a baseline of 175 ± 12 (SE) to a peak of 593 ± 20 impulses/s within 4.6 ± 0.9 s, followed by adaptation, whereas ND declined to 423 ± 20 impulses/s after 1 min. Tissue DA initially increased from a baselineof 17.9 ± 1.2 µM to a peak of 23.2 ± 1.2 µM within 3.0 ± 0.7 s, then declined to 2.6 ± 1.0 µM. The substantialdecrease in tissue DA during normoxic hypercapnia was not consistentwith the parallel changes in DA with ND that were observed for hypoxic stimuli.

     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

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     

High rates of microbial sulphate reduction in a mesophilic ethanol-fed expanded-granular-sludge-blanket reactor

In a mesophilic (30–35 °C), sulphidogenic, ethanol-fed expanded-granular-sludge-blanket reactor, sulphate, at loading rates of up to 10.0–12.0 g Sl⁻¹␣day⁻¹, was removed with an average efficiency of more than 80%. The pH was between 7.7 and 8.3 and the maximal total dissolved sulphide concentration was up to 20 mM S (650 mg S/l). The alkaline pH was maintained by either a pH-control unit with sodium hydroxide or by stripping part of the sulphide and CO₂ from the recycle with nitrogen gas. The superficial upstream liquid velocity (v _up) was 3.0–4.5 m/h. The ratio of ethanol to sulphur was near stoichiometry. At alkaline pH, the activity of the acetotrophic sulphate-reducing bacteria, growing on acetate, was strongly enhanced, whereas at pH below 7.7 the acetotrophic sulphate-reducing bacteria were inhibited by aqueous H₂S. With regard to the removal efficiency and operational stability, external stripping with N₂ and pH control were equally successful. Received: 2 December 1996 / Received revision: 13 March 1997 / Accepted: 15 March 1997     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Effects of ANG II and III and angiotensin receptor blockers on nasal salt gland secretion and arterial blood pressure in conscious Pekin ducks (Anas platyrhynchos)

The vertebrate renin-angiotensin system controls cardiovascular, renal and osmoregulatory functions. Angiotensin II (ANG II) is the most potent hormone of the RAS but in some vertebrate animals angiotensin III (Val⁴-ANG III) may be a hormone. We studied the effects of some angiotensins and mammalian ANG II receptor antagonists on nasal salt gland function and arterial blood pressure in conscious white Pekin ducks. Nasal salt gland fluid secretion (NFS) was induced by a 10 ml · kg⁻¹ bw i.v. injection of a NaCl solution (1000 mosmol · kg⁻¹ H₂O) and maintained by a continuous i.v. infusion of the same solution at a rate of 0.97 ml · min⁻¹. There was a positive linear correlation between nasal fluid [Na⁺] and osmolality, between [Na⁺] and [K⁺], and also between the rate of NFS and [Na⁺] and [K⁺]. [Asp¹,Val⁵]-ANG II (1 nmol · kg⁻¹ i.v.) inhibited NFS but did not change ionic concentrations. Val⁴-ANG III (1 or 5 nmol · kg⁻¹) and ANG I (1-7) (20 nmol · kg⁻¹) had no effect on NFS. [Sar¹, Ile⁸]-ANG II (SARILE) acted as an ANG II receptor agonist and resulted in a prolonged and complete inhibition of NFS. The AT₁ receptor antagonist, losartan (DuP 753) and the AT₂ receptor antagonist, PD 123319 both failed to block the inhibitory effect of [Asp¹, Val⁵]-ANG II on the nasal salt glands. [Asp¹,Val⁵]-ANG II (2 nmol · kg⁻¹ i.v.) increased mean arterial blood pressure (MABP), whereas the same dose of [Asn¹,Val⁵]-ANG II (teleost) had only 30% of the pressor potency of the avian ANG II. Neither 1 nor 5 nmol · kg⁻¹ of Val⁴-ANG III i.v. nor 20 nmol · kg⁻¹ of ANG I (1-7) had any measurable effect on MABP. SARILE blocked completely the pressor response to [Asp¹,Val⁵]-ANG II but the AT₁ antagonists losartan and CGP 48933 and the AT₂ antagonist PD 123319 all failed to block the pressor response to [Asp¹,Val⁵]-ANG II. These results have substantiated an important role of the nasal salt gland in potassium regulation and highlighted a pharmacological dimorphism of saralasin, namely agonist and antagonist to angiotensin II-mediated inhibition of nasal salt gland function and pressor response, respectively. Using specific nonpeptidergic angiotensin II receptor antagonists, we have confirmed the distinct pharmacology of the avian angiotensin II receptors in a nongallinaceous species and the absence of significant angiotensin I (1-7) and angiotensin II effects on the cardiovascular system and nasal salt gland. Accepted: 6 November 1997     

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture

The biodegradation of tributyl phosphate (Bu₃-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu₃-P biodegradation by growing cells is described. Growth at the expense of Bu₃-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu₃-P biodegradation was optimal at 30 °C, reduced at 20 °C and negligible at 4 °C and 37 °C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu₃-P biodegradation significantly, but nitrate had no effect. Sulphate (10–100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 μmol h⁻¹ mg protein⁻¹ and 37 μmol h⁻¹ mg protein⁻¹ respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu₃-P hydrolysis in the presence of 35 mM SO₄ ²⁻. At pH 4.5, 79% of the UO₂ ²⁺ was removed at a flow rate of 1.4 ml/h on a 7-ml test column. Received: 2 June 1997 / Received revision: 15 September 1997 / Accepted: 19 September 1997     

Characteristics of dipeptide transport in pig jejunum in vitro

 Characteristics of dipeptide transport in pig jejunum were investigated in vitro by applying the Ussing-chamber technique and mucosal uptake studies. Addition of both glycyl-l-glutamine and glycyl-l-sarcosine (20 mmol · l⁻¹) to the mucosal buffer solution significantly increased the short-circuit current by 2.60 ± 0.15 and 1.57 ± 0.20 μeq · cm⁻² · h⁻¹, respectively. Concentration-dependent changes in short-circuit current followed Michaelis-Menten kinetics with similar affinity constants for both dipeptides. From unidirectional flux rates for radiolabelled glycyl-l-sarcosine, a net flux rate for glycyl-l-sarcosine of 49.8 ± 6.7 nmol · cm⁻² · h⁻¹ was calculated. In mucosal uptake experiments, the apical influx of ¹⁴C-labelled glycyl-l-sarcosine into isolated porcine mucosa was pH dependent and significantly inhibited by glycyl-l-glutamine. Moreover, RT-PCR studies with primers derived from rabbit PepT1 identified two PCR fragments of identical size to rabbit PepT1 from pig intestinal mRNA preparations. In conclusion, our studies revealed key features of mammalian intestinal peptide transporters and give evidence for a PepT1-like transporter in the pig jejunum that could significantly contribute to the overall amino acid absorption from the gut. Accepted: 30 June 1999     

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