Two-substrate kinetic analysis: a novel approach linking ion and acid-base transport at the gills of freshwater trout,Oncorhynchus mykiss

Summary The novel application of a two-substrate model (Florini and Vestling 1957) from enzymology to transport kinetics at the gills of freshwater trout indicated that Na⁺/acidic equivalent and Cl^-/basic equivalent flux rates are normally limited by the availability of the internal acidic and basic counterions, as well as by external Na⁺ and Cl^- levels. Adult rainbow trout fitted with dorsal aortic and bladder catheters were chronically infused (10–16 h) with isosmotic HCl to induce a persistent metabolic acidosis. Acid-base neutral infusions of isosmotic NaCl and non-infused controls were also performed. Results were compared to previous data on metabolic alkalosis in trout induced by either isosmotic NaHCO₃ infusion or recovery from environmental hyperoxia (Goss and Wood 1990a, b). Metabolic acidosis resulted in a marked stimulation of Na⁺ influx, no change in Cl^- influx, positive Na⁺ balance, negative Cl^- balance, and net H⁺ excretion at the gills. Metabolic alkalosis caused a marked inhibition of Na⁺ influx and stimulation of Cl^- influx, negative Na⁺ balance, positive Cl^- balance, and net H⁺ uptake (=base excretion). Mean gill intracellular pH qualitatively followed extracellular pH. Classical one-substrate Michaelis-Menten analysis of kinetic data indicated that changes in Na⁺ and Cl^- transport during acid-base disturbance are achieved by large increases and decreases in J_max, and by increases in K_m. However, one-substrate analysis considers only external substrate concentration and cannot account for transport limitations by the internal substrate. The kinetic data were fitted successfully to a two-substrate model, using extracellular acid-base data as a measure of internal HCO ₃ ^- and H⁺ availability. This analysis indicated that true J_max values for Na⁺/acidic equivalent and Cl^-/basic equivalent transport are 4–5 times higher than apparent J_max values by one-substrate analysis. Flux rates are limited by the availability of the internal counterions; transport K_m values for HCO ₃ ^- and H⁺ are far above their normal internal concentrations. Therefore, small changes in acid-base status will have large effects on transport rates, and on apparent J_max values, without alterations in the number of transport sites. This system provides an automatic, negative feedback control for clearance or retention of acidic/basic equivalents when acid-base status is changing.Abbreviations Amm total ammonia in water - DMO 55-dimethyl-24-oxyzolidine-dione - J_in unidirectional inward ion movement across the gill - J_out unidirectional outward ion movement across the gill - J_net net transfer of ions (sum of J_in and J_out) across the gill - J_max maximal transport rate for ion - K_m inverse of affinity of transporter for ion - PIO₂ partial pressure of oxygen in inspired water - P_aCO₂ partial pressure of carbon dixide in arterial blood - TAlk titratable alkalinity of the water - PEG polyethylene glycol - NEN New England Nuclear     

Blood-gas,acid-base and catecholamine responses to lactic acid infusion in larval and adult Ambystoma tigrinum

Larval and adult Ambystoma tigrinum were subjected to acidosis by infusing lactic acid (2 M·g^-1) into the peritoneal cavity. Blood was sampled at intervals to establish the time-course of the ensuing acidosis. Both larvae and adults became significantly acidotic after 1 h. The larval acidosis was more pronounced (-4 pH units versus-2 pH units) than adults due to greater extracellular buffering capacity (higher [HCO₃ ^-]) in adults. Both forms recovered in about 8 h. Larvae showed a typical increase in circulating norepinephrine during the initial stages of the acidosis; adults did not, having significantly lower norepinephrine titer than larvae during the acidosis. Both larvae and adults showed transient increases in PO₂ during the acidosis. The ₁ and ₂ antagonists, timolol and butoxamine respectively, (0.2 g·g^-1) were administered to separate groups of larvae. Butoxamine (₂) delayed the recovery from the acidosis by prolonging the increase in arterial PCO₂ and reversing the recovery of [HCO₃ ^-]. Timolol (₁) did not delay recovery. We conclude that ₂ receptors are involved in the catecholamine responses to acidosis in larvae. Catecholamines appear not to play the same role in adult acid-base disturbances as they seem to in larvae.Abbreviations RBC red blood cell     

The effect of glucose on chloride uptake by Chlorella

Active Cl^- uptake by Chlorella fusca was examined by using ³⁶Cl as a label. Under light/air conditions chloride influx from a 2.4·10^-5 M solution was 4.0±0.04 nmol m^-2s^-1. After 70±10 min a stationary 380±40 fold accumulation was reached. In dark/air and dark/argon influx and accumulation were reduced to 25±6%, respectively, 5±1.5% of the light/air control. Cl^- uptake had a broad optimum around pH 7 and showed saturation kinetics with a K _M of 1.25·10^-5 M and a v _max of 7.0 nmol m^-2s^-1 in light/air. Br^- inhibited Cl^- uptake strongly, J^-, ClO ₄ ^- , SO ₄ ^2- , and NO ₃ ^- had no inhibitory effect. Inhibitor studies with carbonyl cyanide m-chlorophenylhydrazone and N,N-dicyclohexylcarbodiimide resulted in a good correlation between Cl^- uptake and ATP level. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and darkness reduced transport activity without affecting the ATP level.The magnitudes of the pH gradient and the membrane potential across the cell membrane were determined and/or estimated under different conditions. It could be shown that in Chlorella Cl^- transport cannot proceed via secondary active H⁺/Cl^- cotransport. In addition, 2H⁺/Cl^- cotransport seems unlikely for energetic reasons. On the basis of the results of this and the following study, a primary active ATP-driven Cl^-/OH^- exchange pump is proposed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhyd razone - DCCD N,N-dicyclohexylcarbodiimide - DCMU 3-(3.4-dichlorophenyl)-1.1-dimethylurea - DMO 5,5-dimethyloxazolidine-2,4-dione - Hepes N-2-hydroxyethylpiperazine-N ethane-sulfonic acid - POPOP 1.4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2.5-diphenyloxazole To whom correspondence should be addressed     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

The physiological responses of freshwater rainbow trout,Oncorhynchus mykiss,during acutely lethal copper exposure

Acutely lethal (24 h) exposure of adult rainbow trout (Oncorhynchus mykiss) to 4.9 mol copper·l^-1 in fresh water (pH 7.9, [Ca²⁺]0.8 mEq·l^-1) caused a rapid decline of plasma Na⁺ and Cl^- and arterial O₂ tension, and initially a pronounced tachycardia. The internal hypoxia probably resulted from histopathologies observed in the gills of fish exposed to copper, such as cell swelling, thickening and curling of the lamellae, and haematomas. Copper cannot therefore be considered purely as an ionoregulatory toxicant during acutely lethal conditions. Mortality during exposure to copper could not simply be explained by the plasma ionic dilution, nor by the internal hypoxia, since arterial O₂ content remained relatively unchanged. Secondary to the ionoregulatory and respiratory disturbances were a number of deleterious physiological responses which included a massive haemoconcentration (haematocrit values as high as 60%) and a doubling of the mean arterial blood pressure. The time-course of these changes suggest that cardiac failure was the final cause of death. In this respect copper exposure resembles low pH exposure in freshwater trout (Milligan and Wood 1982). Copper and H⁺ appear to be similar in both the primary site of their toxic action (the gills) and the secondary physiological consequences which result from acutely lethal exposures. Furthermore, the acute toxicity syndrome observed may be common to many metals which cause ionoregulatory and/or respiratory problems in freshwater fish.Abbreviations C _aO₂ arterial oxygen content - FR water flow rate - Hb haemoglobin - Hct haematocrit - H _m ⁺ net metabolic acid load - IU international unit - MABP mean arterial blood pressure - MCHC mean corpuscular haemoglobin content - MO₂ rate of oxygen consumption - P _aCO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen partial pressure - T _amm total ammonia (=NH₃+NH ₄ ⁺ ) - TCO₂ total carbon dioxide - TOC total organic carbon - %Hb–O₂ percentage of haemoglobin saturated with oxygen     

Differential responses to copper in rainbow trout (Oncorhynchus mykiss) acclimated to sea water and brackish water

Rainbow trout, Oncorhynchus mykiss, acclimated to 33% sea water (12 mg·ml^-1 salinity) experienced significant (10 meq·1^-1) increases in plasma [Na⁺] and [Cl^-] within 5 h of exposure to 6.3 mol copper·1^-1 indicating severe impairment of branchial ionoregulatory capacity. All plasma ion levels subsequently stabilised once the transbranchial [Na⁺] gradient was reduced to zero. The similar ionic strength of the external medium and their body fluids appeared to protect trout maintained in 33% sea water from further ionoregulatory stress and any secondary physiological disturbances during exposure to copper. Despite three- and fourfold greater transbranchial [Na⁺] and [Cl^-] gradients, trout acclimated to full-strength sea water (35 mg·ml^-1 salinity) suffered no major changes in plasma Na⁺, Cl^-, K⁺, or Ca²⁺, blood gases or haematology during 24 h exposure to 6.3 mol copper·1^-1. This reduction in toxicity in full strength sea water cannot be explained by differences in copper speciation. We suggest that during acute exposure to waterborne copper, active NaCl extrusion is unaffected due to the basolateral location of the gill Na⁺/K⁺-ATPase, but that ionoregulatory disturbances can occur due to gill permeability changes secondary to the displacement of surface-bound Ca²⁺. However, in full strength sea water the three-fold higher ambient [Ca²⁺] and [Mg²⁺] appear to be sufficient to prevent any detrimental permeability changes in the presence of 6.3 mol copper·1^-1. Plasma [NH ₊ ⁴ ] and [HCO _- ³ ] were both significantly elevated during exposure to copper, indicating that some aspects of gill ion transport (specifically the apical Na⁺/NH ₊ ⁴ and Cl^-/HCO _- ³ exchanges involved in acid/base regulation and nitrogenous waste excretion) are vulnerable to inhibition in the presence of waterborne copper.Abbreviations C _aO₂ arterial oxygen content - Hb haemoglobin - Hct haematocrit - MABP mean arterial blood pressure - MCHC mean cell haemoglobin content - MO₂ rate of oxygen consumption - P _a CO₂ arterial carbon dioxide tension - P _aO₂ arterial oxygen partial pressure - S salinity - SW sea water - T _Amm total ammonia (=NH₃+NH ₊ ⁴ ) - T _{CO 2} total carbon dioxide - TEP transepithelial potential - TOC total organic carbon - %Hb-O₂ percentage of haemoglobin saturated with oxygen     

Mechanisms of fluid and ion secretion by the parotid gland of the kangaroo,Macropus rufus,assessed by administration of transport-inhibiting drugs

Possible mechanisms of primary fluid formation by macropodine parotid glands were investigated in anaesthetized red kangaroos using ion transport inhibitors. Carotid plasma amiloride concentrations of 0.05–0.5 mmol·l^-1 progressively reduced a stable acetylcholine-evoked half-maximal flow rate of 2.0±0.04 to 0.22±0.024 ml·min^-1 (mean±SEM). Concurrently, saliva bicarbonate concentration and secretion fell (135±1.6 to 67±1.7 mmol·l^-1 and 272±7.6 to 15±2.6 mol·min^-1, respectively); [phosphate], [chloride] and [sodium] rose and [potassium] and osmolality were unaltered. High-rate cholinergic stimulation did not increase saliva flow beyond 11±1.0% of that for equivalent pre-amiloride stimulation. Equipotent levels of amiloride and methazolamide given concurrently were no more effective at blocking flow and bicarbonate secretion than when given separately. Furosemide (up to 2 mmol·l^-1), bumetanide (up to 0.2 mmol·l^-1) and ethacrynate (1 mmol·l^-1) in carotid plasma had no effect on salivary flow or ion concentrations. During methazolamide blockade, furosemide did not curtail the concurrent increase in salivary [chloride]. Chlorothiazide at 0.25–1.0 mmol·l^-1 caused progressive depression of saliva flow and [bicarbonate], and elevation of [chloride]. 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid at 0.1 mmol·l^-1 was without effect, whereas at 0.5 mmol·l^-1 it stimulated fluid secretion and increased saliva [protein], [sodium], [potassium], [bicarbonate] and osmolality. Concurrently, mean arterial blood pressure and pulse pressure fell and heart rate, haematocrit and carotid artery plasma flow rose. These responses were absent if saliva flow was kept constant by reduction in cholinergic stimulation during 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid administration. It is concluded that secretion of primary fluid by the kangaroo parotid is initiated mainly (>90%) by secretion of bicarbonate which is formed in the endpiece cells from CO₂ delivered by the circulation. No evidence was found for initiation of fluid secretion by chloride transport involving basolateral Na⁺-K⁺-2Cl^- symports, Na⁺-Cl^- symports or Cl^-/HCO ₃ ^- antiports.Abbreviations CA carbonic anhydrase - CAI carbonic anhydrase inhibitors - MAP mean arterial blood pressure - PAH p-aminohippurate - SITS 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid     

The effects of the adrenoreceptor agonists phenylephrine and isoproterenol on the intracellular ion concentrations of branchial epithelial cells of brown trout (Salmo trutta L.)

Brown trout were fitted with indwelling, intraperitoneal catheters and injected with 4–6 mol · kg^-1 of the -receptor agonist phenylephrine or the -receptor agonist isoproterenol. The intracellular concentrations of sodium, chlorine, potassium and phosphorus in the pavement epithelial cells and the mitochondria-rich cells of the branchial epithelium were measured by X-ray microanalysis 1 h after the injection of the adrenoreceptor agonists. Injection with phenylephrine resulted in a significant increase in intracellular chlorine and potassium in mitochondria-rich cells and a significant but relatively smaller increase in chlorine in pavement epithelial cells. Injection with isoproterenol resulted in a significant increase in sodium and chlorine concentration in pavement epithelial cells and a significant decrease in potassium concentration. The only significant effect of isoproterenol injection on mitochondria-rich cells was a decrease in intracellular chlorine concentration. The results suggest that these adrenoreceptor agonists have a direct effect on the influx of Na⁺ and Cl^- across the branchial epithelium. These effects may be a mechanism for acid-base regulation during the severe stress conditions that elicit catecholamine release in vivo. These results corroborate previous studies using X-ray microanalysis which suggested that pavement epithelial cells are the sites of Na⁺ uptake in freshwater fish whilst Cl^- uptake occurs via mitochondria-rich cells.Abbreviations LTSEM low-temperature scanning electron microscope - MR cells mitochondria-rich cells - PE cells pavement epithelial cells - XRMA X-ray microanalysis     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume     

The effects of experimentally altered gill chloride cell surface area on acid-base regulation in rainbow trout during metabolic alkalosis

To evaluate the role of the gill chloride cells in regulating metabolic alkalosis in rainbow trout (Oncorhynchus mykiss), the surface area of branchial chloride cells was altered experimentally using combined cortisol/ovine growth hormone injections. Long-term (10-day) treatment of fish with cortisol/ovine growth hormone caused an increase in the two-dimensional chloride cell fractional surface area when compared to uninjected fish (from 8.4 to 29.7%). This was the combined result of an increase in the size of individual cells (from 34.6 to 59.2 m²) and increased numbers of cells (from 2368 to 5006 cells · mm^-2). Metabolic alkalosis was induced by intra-arterial infusion of 140 mmol · l^-1 NaHCO₃; control fish were infused with 140 mmol · l^-1 NaCl. Blood pH and plasma [HCO₃ ^-] increased in both the untreated and the cortisol/ovine growth hormone-treated fish. However, the increases in pH (from 8.05 to 8.53) and [HCO₃ ^-] (from 5.9 to 22.2 mmol · l^-1) in the untreated fish were significantly greater than in the cortisol/ovine growth hormone-treated fish (pH increased from 7.78 to 8.11; [HCO₃ ^-] increased from 5.5 to 13.9 mmol · l^-1). In all fish, NaHCO₃ infusion elicited an increase in the rate of branchial basic equivalent excretion (acidic equivalent uptake) which, in turn, was caused by decreases and increases in branchial Na⁺ uptake and Cl^- uptake, respectively. In the untreated fish, there was a pronounced increase (75%) in chloride cell surface area during NaHCO₃ infusion. The attenuation of the metabolic alkalosis during HCO₃ ^- infusion in the cortical/ovine growth hormone-treated fish was caused, at least in part, by an enhancement of branchial basic equivalent excretion. In these fish that already displayed a proliferation of chloride cells, there was no further increase in chloride cell surface area. The changes in Na⁺ influx and Cl^- influx were quantitatively similar during NaHCO₃ infusion in both groups. This suggests that the greater rate of base excretion in the cortisol/ovine growth hormone-treated fish was caused by a greater percentage of Cl^- uptake being coupled to HCO₃ ^- excretion and less to Cl^- excretion (Cl^- exchange diffusion).Abbreviations Amm total ammonia - bw body weight - CC chloride cell - CCFA chloride cell fractional area - cort/oGH cortisol/ovine growth hormone - dpm disintegrations per minute - J ^Amm net flux of total ammonia - J _in unidirectional influx - J _inCl^- chloride ion uptake - J _inNa⁺ sodium ion uptake - J _netH⁺ net acidic equivalent flux - J ^TA net flux of titrable alkalinity - MS 222 ethyl-m-aminobenzoate - oGH ovine growth hormone - PVC pavement cell - SEM scanning electron microscope - TA titrable alkalinity     

Acid-base and electrolyte regulation,and haemolymph gas transport in crayfish,Astacus astacus,exposed to soft,acid water with and without aluminium

Summary Intermoult crayfish (Astacus astacus) were exposed to acid (pH 4), soft water ([Ca⁺⁺]=100 mol·l^–1) in the absence and presence of aluminium (25 mol·l^–1) for variable time periods (up to 21 days) in order to assess the consequences for acid-base and electrolyte balance and haemolymph gas transport. Haemolymph osmolality and concentration of major ions decreased drastically and to a similar extent in acid and acid-aluminium water. Muscle tissue ion concentrations were, however, regulated at an almost constant level. A severe metabolic acidosis was gradually developed, attaining a haemolymph metabolic acid load of 6–7 mequiv·l^–1 after 12–21 days. The acidosis was partially compensated by ventilatory means, with the postbranchial haemolymph PCO₂ decreasing earlier in acidaluminium-exposed than in acid-exposed specimens. Hyperventilation seemed to be a direct acid-base regulatory response, since the rise in postbranchial PCO₂ had only minimal influence on haemolymph O₂ transport. The Bohr effect of Astacus astacus haemocyanin was low (log P₅₀/GdpH=-0.24), and the mean P₅₀ only increased from 15 to 19 mmHg after 21 days of acid exposure. The decrease in O₂ affinity with decreasing pH was accompanied by a decrease in the cooperativity of O₂ binding. The haemolymph haemocyanin concentration was not affected by acid and acid-aluminium exposure, but decreased after 21 days due to starvation. Muscle tissue aluminium concentrations were unaffected, whereas gill tissue concentrations increased in acid-aluminium exposed crayfish, most likely due to accumulation of aluminium on the gill surface. Mortality was low, and an internal hypoxia and lactacidosis was not developed in either of the experimental groups. This suggests that the gas transfer qualities of the chitincovered gills of crayfish are much less sensitive to acid and acid-aluminium stress than the gills of teleost fish.Abbreviations Hc haemocyanin - SO₂ saturation of Hc with O₂ - P ₅₀ oxygen tension of haemolymph at 50% SO₂ - n ₅₀ Hills coelficient around 50% SO₂     

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl^– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^–1), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol·l^–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l^–1 as NH₄Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO ₃ ^– buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol·l^–1). Neither bafilomycin A₁ (3 mol·l^–1) nor Cl removal altered the intracellular pH recovery rate. The K _m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol·l^–1, and the rate constant at V _max was 0.008·s^–1 (equivalent to 5.60 mmol H⁺·l^–1 cell water·min^–1), which was achieved at external Na⁺ levels from 25 to 140 mmol·l^–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO ₃ ^– -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺-ATPase.Abbreviations BCECF 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein - BCECF/AM 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester - Cholin-Cl choline chloride - DMSO dimethyl sulfoxide - EDTA ethylene diamine tetra-acetic acid - FBS foetal bovine serum - H ⁺ -ATPase Proton-dependent adenosine triphosphatase - HEPES N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid] - pH _i intracellular pH - pH _e extracellular pH - PBS phosphate-buffered saline - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid     

Some characteristics of anion transport at the tonoplast of oat roots,determined from the effects of anions on pyrophosphatedependent proton transport

The effects of anions on inorganicpyrophosphate-dependent H⁺-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H⁺-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl^-, Br^- and NO₃-) stimulated H⁺-transport while others (malate, and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H⁺-transport by Cl^- showed saturation kinetics whereas that by NO₃- consisted of both a saturable component and a linear phase. For Cl^- and NO₃-, the saturable phase had a K _m of about 2 mol·m^-3. The anions that stimulated H⁺-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H⁺-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP 1,3-bis[tris(hydroxymethyl)-methylamino]-propane - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - IDA iminodiacetate - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi morganic pyrophosphate     

The effects of amiloride on electrolyte transport and acid-base balance in the larval salamander,Ambystoma tigrinum

Summary The responses of net and unidirectional fluxes of Na⁺ and acid-base balance to the drug amiloride were assessed during normocapnia and hypercapnia in larval salamanders, Ambystoma tigrinum. Isotope flux measurements demonstrated that 10^-4 M amiloride in the external medium inhibits Na⁺ influx during normocapnia and reverses the usual increase in influx of this ion during hypercapnia, causing a significant decrease instead. Measurements of blood-gas/acid-base balance conditions of artcrially cannulated salamanders demonstrated a significant metabolic acidosis in amiloridetreated animals that did not occur in untreated animals over the same period. the same concentration of amiloride also blocked the normal compensatory increase in [HCO _- ³ ] that follows a respiratory acidosis produced by a hypercapnic environment.Abbreviations IU international nnits - J _in influx - J _net net flux - PCO ₂ parial pressure of carbon dioxide     

Characterization of the Na+-dependent taurine influx in flounder erythrocytes

About 92% of the taurine influx in flounder erythrocytes at physiological conditions in vitro (330 mosmol·l^-1, 145 mmol·l^-1 Na⁺, 0.30 mmol·l^-1 taurine) is Na⁺-dependent. This influx is highly specific for taurine. The -amino compounds hypotaurine and -alanine were the only compounds which mimicked the inhibitory effect of taurine on influx of [¹⁴C]taurine, the former more than the latter. Counterexchange of taurine was also mediated by the taurine transporters. Reduction of osmolality per se did not affect the activity of these transporters. Non-linear regression analysis of the influx values revealed the presence of two different influx systems: a system with high affinity and low capacity and another with low affinity and high capacity. However, we cannot exclude the possibility that this influx of taurine was mediated by only one transporter which operated in different modes depending on the extracellular Na⁺ concentration. On the assumption that the Na⁺-dependent influx was mediated by two separate systems, the maximal velocity of the low capacity system was 2.55 nmol·g dry weight^-1·min^-1 at 145 mmol·ll^-1 extracellular Na⁺. This capacity was about 50% lower than that of the high capacity system. The Michaelis constants were 0.013 and 1.34 mmol·l^-1, respectively. Reduction of the extracellular Na⁺ concentration reduced maximal velocity and the affinity to taurine of both transport systems. At 10 mmol·l^-1 Na⁺ or lower concentrations the high capacity system did not seem to operate. The activation method suggested that each taurine molecule transported by the high capacity system was accompanied by two Na⁺. The stoichiometry of the low capacity system was 1 taurine: 1 Na⁺. The Hill-coefficient for both transport systems was 1.00.Abbreviations cpm counts per minute - dw dry weight - GABA -amino-n-butyric acid - K _m Michaelis constant - pK _b basic dissociation constant - SD standard deviation - -ABA Dl--amino-n-butyric acid - V _max maximal velocity - ww wet weight     

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K−Cl cotransport

Pathways of K⁺ movement across the erythrocyte membrane of frog Rana temporaria were studied using ⁸⁶Rb as a tracer. The K⁺ influx was significantly blocked by 0.1 mmol·l^-1 ouabain (by 30%) and 1 mmol·l^-1 furosemide (by 56%) in the red cells incubated in saline at physiological K⁺ concentration (2.7 mmol·l^-1). Ouabain and furosemide had an additive effect on K⁺ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K⁺ influx saturated as f(K⁺)_e with apparent K _m values for external K _e ⁺ concentration of 0.96±0.11 and 4.6±0.5 mmol·l^-1 and V _max of 0.89±0.04 and 2.8±0.4 mmol·l cells^-1·h^-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of K _e ⁺ medium concentration. Total K⁺ influx was significantly reduced when frog erythrocytes were incubated in NO _- ³ medium. Furosemide did not affect K⁺ transport in frog red cells in NO ₃ ^- media. At the same K _e ⁺ concentration the ouabain-furosemide-insensitive K⁺ influx in Cl^- medium was significantly greater than that in NO _- ³ medium. We found no inhibitory effect of 1 mmol·l^-1 furosemide on Na⁺ influx in frog red cells in Cl^- medium. K⁺ loss from the frog erythrocytes in a K⁺-free medium was significantly reduced (mean 58%) after replacement of Cl^- with NO _- ³ . Furosemide (0.5 mmol·l^-1) did not produce any significant reduction in the K⁺ loss in both media. The Cl^--dependent component of K⁺ loss from frog red cells was 5.7±1.2 mmol·l^-1·h^-1. These results indicate that about two-thirds of the total K⁺ influx in frog erythrocytes is mediated by a K–Cl cotransport which is only partially blocked by furosemide.Abbreviations DMSO dimethyl sulphoxide - K _e ⁺ external concentration of K⁺ - K _m apparent Michaelis constant for external - K⁺ K _e ⁺ at V _max/2 - RBC red blood cell(s) - V _max maximal velocity of the unidirectional K⁺ influx - TRIS tris(hydroxymethyl)aminomethane     

Secretion of electrolytes,protein and urea by the mandibular gland of the common wombat (Vombatus ursinus)

Saliva was collected from the mandibular glands of anaesthetized common wombats (Vombatus ursinus) to ascertain maximal flow rates, salivary compostion and possible adaptations, particularly PO₄ ^3- secretion, to assist digestion. After temporary catheterization of the main duct through its oral opening, salivary secretion was evoked at flow rates ranging from 0.02±0.002 (±SEM) ml·min^-1 (0.7±0.07 l·min^-1·kg body weight^-1) to 0.4±0.05 ml·min^-1(14±1.9 l·min^-1·kg body weight^-1) by ipsilateral intracarotid infusion of acetylcholine. The [Na⁺] (15±5.1 to 58±8.6 mmol·l^-1) and [HCO₃ ^-] (35±1.9 to 60±1.9 mmol·l^-1) were positively correlated with salivary flow rate. The [K⁺] (58±5.2 to 30±2.4 mmol·l^-1), [Ca²⁺] (10.4±1.67 to 4.1±0.44 mmol·l^-1), [Mg²⁺] (0.94±0.137 to 0.17±0.032 mmol·l^-1), [Cl^-] (71±9.2 to 45±6.0 mmol·l^-1), [urea] (9.3±0.79 to 5.1±0.54 mmol·l^-1), H⁺ activity (29±1.6 to 17±1.6 nEq·l^-1) and amylase activity (251±57.4 to 92±23.3 kat·l^-1) were negatively correlated with flow. Both concentration and osmolality fell with increasing flow at the lower end of the flow range but osmolality always increased again by maximal flow whereas the relation between protein and flow was not consistent at the higher levels of flow and stimulation. Salivary [PO₄ ³⁺] was not correlated with flow and at 3–14% of the plasma concentration was extremely low. Thus, in contrast to its nearest relative, the koala (Phascolarctos cinereus), the wombat secretes little PO₄ ³⁺ presumably because it does not need high levels of PO₄ ³⁺ in its saliva to facilitate microbial digestion of plant fibre.Abbreviations bw body weight - ww wet weight     

Acquisition and assimilation of gaseous ammonia as revealed by intracellular pH changes in leaves of higher plants

Atmospheric ammonia (NH₃) from various anthropogenic sources has become a serious problem for natural vegetation. Ammonia not only causes changes in plant nitrogen metabolism, but also affects the acid-base balance of plants. Using the pH-sensitive fluorescent dyes pyranine and esculin, cytosolic and vacuolar pH changes were measured in leaves of C₃ and C₄ plants exposed for brief periods to concentrations of NH₃ in air ranging from 1.33 to 8.29 mol NH₃ · mol^-1 gas (0.94–5.86 mg · m^-3). After a lag phase, uptake of NH₃ from air at a rate of 200 nmol NH₃ · m ^{- 2} leaf area · s^{- 1} into leaves of Zea mays L. increased pyranine fluorescence indicating cytosolic alkalinisation. The increase was much larger in the dark than in the light. In illuminated leaves of the C₃ plant Pelargonium zonale L. and the C₄ plants Z. mays and Amaranthus caudatus L., NH₃-dependent cytosolic alkalinisation was particularly pronounced when CO₂ was supplied at very low levels (16 or 20 mol CO₂ · mol^{- 1} gas, containing 210 mmol O₂ · mol^{- 1} gas). An increase in esculin fluorescence, which was smaller than that of pyranine, was indicative of trapping of some of the NH₃ in the vacuoles of leaves of Spinacia oleracea L. and Z. mays. Photosynthesis and transpiration remained unchanged during exposure of illuminated leaves to NH₃, yielding an influx of 200 nmol NH₃ · m^-2 leaf area · s^-1 for up to 30 min, the longest exposure time used. Both CO₂ and O₂ influenced the extent of cytosolic alkalinisation. At 500 mol CO₂ · mol^-1 gas the cytosolic alkalinisation was suppressed more than at 16 or 20 mol CO₂ · mol^-1 gas. The suppressing effect of CO₂ on the NH₃induced alkalinisation was larger in illuminated leaves of the C₄ plants Z. mays and A. caudatus than in leaves of the C₃ plant P. zonale. A reduction of the O₂ concentration from 210 to 10 mmol O₂ · mol ^-1 gas, which inhibits photorespiration, increased the NH₃induced cytosolic alkalinisation in C₃ plants. Suppression by CO₂ or O₂ of the alkaline pH shift caused by the dissolution and protonation of NH₃ in queous leaf compartments, and possibly by the production of organic compounds synthesised from atmospheric NH₃, indicates that NH₃ which enters leaves is rapidly assimilated if photosynthesis or photorespiration provide nitrogen acceptor molecules.This work was supported by the Biotechnology and Biological Sciences Research Council and the Deutsche Forschungsgemein-schaft within the framework of the research of Sonderforschun-gsbreich 251 of the University of Würzburg. We are grateful to Dr. B. Wollenweber (The Royal Veterinary and Agricultural University, Denmark) for discussions.     

The relationship of the charasome to chloride uptake in Chara corallina: physiological and histochemical investigations

A possible role of the charasome in terms of chloride transport into Chara corallina Klein ex. Willd., em. R.D.W. is examined. The branches of Chara contain the most charasome material and are shown to be very effective in acquiring Cl^- to support continued shoot growth. The early maturation of the branches, the rather large Cl^- fluxes into these cells, and their ability of translocate Cl^- to growing cells of the shoot indicate a special role of these branches in Cl^- accumulation. The structure of the charasome, with its extensive periplasmic space, appears especially suited as a site for H⁺–Cl^- cotransport (influx). We show, by histochemical assay, that the charasomes of mature cells contain ATPase activity; such activity is absent in growing charasomes of very young cells. ATPase activity is also associated with the plasmodesmata of C. corallina. Charasome ATPase activity and Cl^- uptake are both inhibited by p-chloromercuribenzenesulfonic acid (1 mM) or diethylstibestrol (40 M; 45 min). The anion transport inhibitor, 4,4-diisothiocyano-2,2-disulfonic acid stilbene (1 mM) had no effect on Cl^- transport and inhibited ATPase activity only when applied after chemical fixation of the cells. Results of an attempt to demonstrate the presence of Cl^- within the cytoplasmic tubules of the charasome, using a silver precipitation technique, proved difficult to interpret because of a reaction between the silver and a cellular substance produced in the light.Abbreviations CPW Chara pond water - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-disulfonic acid stilbene - Mops 3-(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine