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     

Isolation and characterization of thiosulfate reductases from the green alga Chlorella fusca

Thiosulfate-reductase activity (TSR) measured as sulfide release from thiosulfate was detected in crude extracts of Chlorella using dithioerythritol (DTE) as electron donor. Purification of this activity by ammonium-sulfate precipitation between 35% and 80% followed by Sephadex G-50 gel filtration, diethylaminoethyl-cellulose chromatography, and gel filtration on Biogel A 1.5 M led to four distinct proteins having molecular weights of: TSR I, 28000; TSR II, 26500; TSR III_a, 55000; TSR III_b, 24000 daltons. These thiosulfate reductases were most active with DTE; the monothiols glutathione, l-cysteine, and -mercaptoethanol had little activity towards this system. The following pH optima were obtained: for TSR I and TSR II, 9.0; for TSR III_a, 8.5; and for TSR III_b, 9.5. The apparent-K_m data for DTE and thiosulfate were determined to: TSR I, 0.164 mmol·l^-1 and TSR II, 0.156 mmol·l^-1; K_mDTE TSR I, 1.54 mmol·l^-1 and TSR II 1.54 mmol·l^-1. The thiosulfate reductases III_a and III_b were further stimulated by addition of thioredoxin. All TSR fractions catalyzed SCN formation from thiosulfate and cyanate and thus had rhodanese activity; this activity, however, could only be detected in the presence of thiols.Abbreviations DTE dithioerythritol - TSR thiosulfate reductase Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

The effect of carbonic anhydrase inhibitors on secretion by the parotid and mandibular glands of red kangaroos Macropus rufus

Summary The effects of carbonic anhydrase inhibitors on secretion by macropodine parotid and mandibular glands were investigated using anaesthetized red kangaroos. In the parotid gland, acetazolamide (500 mol·l^-1) reduced a stable acetylcholine-evoked, half-maximal flow rate of 2.02±0.034 to 0.27±0.023 ml·min^-1 (87% reduction). Concurrently, salivary bicarbonate concentration and secretion fell (129.4±1.46 to 80.9±1.63 mmol·l^-1 and 264.8±7.96 to 22.3±2.30 mol·min^-1, respectively), phosphate and chloride concentrations rose (14.0±0.79 to 27.6±0.85 mmol·l^-1 and 5.6±0.25 to 27.5±1.32 mmol·l^-1, respectively), sodium concentration and osmolality were unaltered, and potassium concentration fell (8.8±0.33 to 6.4±0.29 mmol·l^-1). High-rate cholinergic stimulation during acetazolamide blockade was unable to increase salivary flow beyond 11±0.9% of that for equivalent unblocked control stimulation. However, superimposition of isoprenaline infusion on the acetylcholine stimulation caused a three-fold increase in the blocked flow rate. These treatments were accompanied by small increases in salivary phosphate and chloride concentrations but not bicarbonate concentration. Methazolamide infusion caused similar changes in parotid secretion. In the mandibular gland, acetazolamide infusion had no effect on salivary flow rate during either low- or high-level acetylcholine stimulation. Acetazolamide caused no alterrations in salivary electrolyte secretion at low flow rates, but curtailed the rise in bicarbonate concentration associated with high-level acetylcholine stimulation. Acetazolamide administration did not affect the increase in salivary flow rate associated with isoprenaline infusion, but did block the concomitant increase in bicarbonate concentration and secretion substantially. It was concluded that neither cholinergic nor adrenergic stimulation of mandibular fluid secretion depends on secretion of bicarbonate derived from catalysed hydration of CO₂, but a substantial proportion of the increase in bicarbonate secretion during isoprenaline administration, which is probably ductal in origin, is so dependent. In contrast to other salivary glands, including the ovine parotid, fluid secretion by the kangaroo parotid gland during cholinergic stimulation is largely dependent (about 90%) on secretion of bicarbonate derived from hydration of CO₂ catalysed by glandular carbonic anhydrase. Fluid secretion during adrenergic stimulation is not bicarbonate dependent.Abbreviations b.w. body weight - PAH p-aminohippurate - PCO₂ partial pressure carbon dioxide - PCO₂ partial pressure of 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     

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     

Ion transport across leech integument

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5±0.5 k· cm^-2) with an initial short-circuit current of 29.0±2.9 A·cm^-2. Removal of Na⁺ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50mol·l^-1) added to the basolateral solution, depressed the short-circuit current completely. The Na⁺ current saturated at a concentration of 90 mmol Na⁺·l^-1 in the apical solution (K _M=11.2±1.8 mmol·l^-1). Amiloride (100 mol·l^-1) on the apical side inhibited ca. 40% of the Na⁺ current and indicated the presence of Na⁺ channels. The dependence of Na⁺ current on the amiloride concentration followed Michaclis-Menten kinetics (K _i=2.9±0.4 mol·l^-1). The amiloride analogue benzamil had a higher affinity to the Na⁺ channel (K _i=0.7±0.2 mol·l^-1). Thus, Na⁺ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na⁺·l^-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8±2.3% of total short-circuit current. Higher Na⁺ concentrations lead to a decrease in amiloride-blockade short-circuit current. Sitmulation of the tissue with cyclic adenosine monophosphate (100 mol·l^-1) and isobutylmethylxanthine (1 mmol·l^-1) nearly doubled short-circuit current and increased amiloride-sensitive Na⁺ currents by 50%. By current fluctuation analysis we estimated single Na⁺ channel current (2.7±0.9 pA) and Na⁺ channel density (3.6±0.6 channels·m^-2) under control conditions. After cyclic adenosine monophosphate stimulation Na⁺ channel density increased to 5.4±1.1 channels·m^-2, whereas single Na⁺ channel current showed no significant change (1.9±0.2 pA). These data present a detailed investigation of an invertebrate epithelial Na⁺ channel, and show the similarities and differences to vertebrate Na⁺ channels. Whereas the channel properties are different from the classical vertebrate Na⁺ channel, the regulation by cyclic adenosine monophosphate seems similar. Stimulation of Na⁺ uptake by cyclic adenosine monophosphate is mediated by an increasing number of Na⁺ channels.Abbreviations slope of the background noise component - ADH antidiuretic hormone - cAMP cyclic adenosine monophosphate - f frequency - f _c coner frequency of the Lorentzian noise component - Hepes N-hydroxyethylpiperazine-N-ethanesulphonic acid - BMX isobutyl-methylxanthine - i _Na single Na⁺ channel current - I _Na max, maximal inhibitable Na⁺ current - I _SC short circuit current - K _i half maximal blocker concentration - K _M Michaelis constandard error of the mean - S _(f) power density of the Lorentzian noise component - S ₀ plateau value of the Lorentzian noise component - TMA tetramethylammonium - Trizma TRIS-hydroxymethyl-amino-methane - V _max maximal reaction velocity - V _T transepithelial potential - K half maximal blocker concentration     

Thermal behavior of round and wagtail dancing honeybees

The thermal behavior of round and wagtail dancing honeybees (Apis mellifera carnica) gathering sucrose solutions of concentrations between 0.5 and 2 mol·l^-1 was investigated under field conditions by infrared thermography (30–506 m flight distance). During the stay inside the hive thoracic surface temperature ranged from 31.4 to 43.9 °C. In both round and wagtail dancing honeybees the concentration of sucrose in the food influenced dancing temperature in a non-linear way. Average dancing temperature was 37.9 °C in foragers gathering a 0.5 mol·l^-1 sucrose solution, 40.1°C with a 1 mol·l^-1, 40.6°C with a 1.5 mol·l^-1 and 40.7°C with a 2 mol·l^-1 solution. The variability of thoracic temperature was highest with the 0.5 mol·l^-1 and lowest with the 1.5 and 2 mol·l^-1 concentrations. Thoracic temperatures during trophallactic contact with hive bees were similar to dancing temperature at 1.5 mol·l^-1 but lower at the other concentrations. During periods of distribution of food to hive bees (trophallactic contact >2.5s) the dancers' thorax cooled down by more than 0.5°C considerably more frequently with the 0.5 mol·l^-1 solution (65% of cases) than with the 1.5 mol·l^-1 solution (26%). By contrast, heating the thorax up by more than 0.5°C was infrequent with the 0.5 mol·l^-1 solution (2%) but occurred at a maximum rate of 26% with the 1.5 mol·l^-1 solution. Bees gathering the 1 or 2 mol·l^-1 solutions showed intermediate behavior. Linear model analysis showed that at higher concentrations the dancers compensated better for variations of hive air temperature: per 1 °C increase of hive temperature dancing temperature increased by 0.34, 0.22, 0.12, and 0.13 °C with 0.5, 1, 1.5, and 2 mol·l^-1 sucrose solutions, respectively. The results furnish evidence that dancing honeybees follow a strategy of selective heterothermy by tuning their thermal behavior to the needs of the behavior performed at the moment. Thoracic temperature is regulated to a high level and more accurately when fast exploitation of profitable food sources is recommended. Thoracic temperature is lowered when the ratio of gain to costs of foraging becomes more unfavorable.Abbreviations SD standard deviation - SD _reg SD around regression line - H _rel relative humidity at feeding station - T _a air temperature at feeding station - T _i air temperature near the dancers - T _d Thoracic surface temperatures - T _d dancing - T _tr trophallactic contact (distribution of food) - T _w walking - T _stay mean temperature of total stay in the hive     

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     

Anoxia and freezing exposures stimulate covalent modification of enzymes of carbohydrate metabolism in Littorina littorea

The effects of anoxia (N₂ atmosphere at 5 °C) or freezing (at-8 °C) exposure in vivo on the activities of five enzymes of carbohydrate metabolism were assessed in foot muscle and hepatopancreases of the marine periwinkle Littorina littorea. Changes in glycogen phosphorylase, glycogen synthetase, pyruvate kinase and pyruvate dehydrogenase under either stress were generally consistent with covalent modification of the enzymes to decrease enzyme activity and/or convert the enzyme to a less active form. However, no evidence for a similar covalent modification of phosphofructokinase was found. The metabolic effects of freezing and anoxia were generally similar, suggesting that a primary contributor to freezing survival is the implementation of anaerobic metabolism and metabolic arrest mechanisms that also promote anoxia survival in marine molluses. However, in hepatopancreas phosphorylase was activated and pyruvate kinase remained in two enzyme forms in freezing-exposed snails, contrary to the results for anoxic animals. Ion exchange chromatography on DE-52 Sephadex revealed the presence of two forms of pyruvate kinase in both tissues of control L. littorea, eluting at 30–50 mmol·1^-1 KCl (peak I) or 90–110 mmol·1^-1 KCl (peak II). Anoxia exposure converted pyruvate kinase in both tissues to the peak I form, as did freezing for foot muscle pyruvate kinase. Kinetic analysis showed that peak I pyruvate kinase had lower affinities for substrates, phosphoenolpyruvate and ADP, and was very strongly inhibited by l-alanine compared with the peak II enzyme. Peak I pyruvate kinase had an I ₅₀ value for l-alanine of 0.38 mmol·1^-1, whereas peak II pyruvate kinase was unaffected by l-alanine evenat 40 mmol·1^-1. In vitro incubation of extracts from control foot muscle under conditions promoting phosphorylation or dephosphorylation identified the peak I and II forms as the low and high phosphate forms, respectively. This result for L. littorea pyruvate kinase was highly unusual and contrary to the typical effect of anoxia on pyruvate kinase in marine molluscs which is to stimulate the phosphorylation of pyruvate kinase and, thereby, convert the enzyme to a less active form.Abbreviations AABS p-(p-aminophenylazo)benzene sulphonic acid - F2, 6P fructose-2,6-bisphosphate - F6P fructose-6-phosphate - G6P glucose-6-phosphate - GP glycogen phosphorylase - GS glycogen synthase - I ₅₀ inhibitor concentration reducing enzyme velocity by 50% - MR metabolic rate - PDH pyruvate dehydrogenase - PEP phosphoenopyruvate - PFK phosphofructokinase - PK pyruvate kinase - SW sea water - F _a air temperature - TCA trichloroacetic acid - UDPG uridine-diphosphate glucose - WW wet weight     

Regulation of pH in the isolated perfused gills of the shore crabCarcinus maenas

Isolated posterior gills (no. 7) of shore crabsCarcinus maenas acclimated to brackish water of a salinity of 10 S were bathed and perfused with 50% sea water (200 mmol·l^-1 Na⁺), and the internal perfusate collected during subsequent periods of 5 min. During a single passage through the gill the pH of the perfusion medium decreased from ca. 8.1 to ca. 7.7, a result implying that the gill possesses structures which recognize unphysiologically high pH values in the haemolymph and regulates them down to physiological values of ca. 7.7. The calculated apparent proton fluxes from the epithelial cells into the haemolymph space amounted to 17.9 mol·g fw^-1·h^-1, a value of only 3.8% of net Na⁺ fluxes observed under comparable conditions. When 0.1 mmol·l^-1 KCN, an inhibitor of mitochondrial cytochrome oxidase, or 5 mmol·l^-1 ouabain, a specific inhibitor of Na⁺/K⁺-ATPase were applied in the internal perfusate, down-regulation of pH was no longer observed and the gill was completely depolarized, i.e. transepithelial potential differences dropped from-7.8 to 0 mV (haemolymph space negative to bath). Regulation of pH was completely inhibited by antagonists of carbonic anhydrase (0.1 mmol·l^-1 acetazolamide or 0.01 mmol·l^-1 ethoxyzolamide) applied in the perfusate. Inhibitors of Na⁺/H⁺ exchange, 0.1 mmol·l^-1 amiloride applied in the external bathing medium or in the internal perfusate, and symmetrical 0.01 mmol·l^-1 5-(N-ethyl-N-isopropyl)amiloride, as well as inhibitors of Cl^-/HCO₃ ^- exchange and Na⁺/HCO₃ ^- cotransport, 0.5 mmol·l^-1 4,4-diisothiocyanatostilbene-2,2-disulphonate or 0.3 mmol·l^-1 4-acetamido-4-isothiocyanatostilbene 2,2-disulphonate applied on both sides of the gill, and inhibitors of H⁺-ATPase, 0.05 mmol·l^-1 N-ethylmaleimide and 0.1 mmol·l^-1 N,N-dicyclohexylcarbodiimide —applied on both sides of the gill — did not alter the acidification of the perfusate observed in controls. Using artificial salines buffered to pH 8.1 with 0.75 mmol·l^-1 tris (hydroxymethyl) aminomethane instead of 2 mmol·l^-1 HCO₃ ^-, apparent proton fluxes were reduced to 11% of controls, a result suggesting that pH regulation by crab gills needs the presence of HCO₃ ^-. The findings obtained suggest that pH regulation by crab gills depends on the oxidative metabolism of the intact branchial epithelium and that carbonic anhydrase plays a central role in this process. Na⁺/H⁺ exchange, anion exchange or cotransport and active proton secretion seem not to be involved. While unimpaired active ion uptake is a prerequisite for pH regulation, ion transport itself is independent of it.Abbreviations acetazolamide (N-[sulphamoyl-1, 3, 4-thiadiazol-2-yl]-acetamide) - amiloride 3,5-diamino-6-chloropyrazinoyl-guanidine - CA carbonic anhydrase - DBI dextrane-bound inhibitor thiadiazolesulphonamide - DCCD N N dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanato-stilbene-2,2-disulphonate - EIPA 5-(N-ethyl-N-isopropyl) amiloride - ethoxyzolamide 6-ethoxy-2-benzothiazole-sulphonamide - fw fresh weight - J _H ⁺ apparent proton flux - NEM N-ethylmaleimide - PD transepithelial potential difference - PEG-STZ polyethylene-glycol-thiadiazolesulphonamide - STTS 4-acetamido-4-isothiocyanatostibene 2,2-disulphonate - SW sea water - TRIS tris(hydroxymethyl)aminomethane     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pH_i) and luminal pH (pH_lu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH_o 6.9). In proximal segments both pH_i (7.43±0.20) and pH_lu (7.10±0.24) were significantly lower than in distal segments (pH_i 7.70±0.29, pH_lu 8.09±0.15). Steady-state pH_i of proximal segments was much less sensitive to changes in pH_o than pH of the luminal fluid (pH_lu/pH_o was 0.49 while pH_i/pH_o was 0.18; pH_o 6.50–7.20). Re-alkaliniziation from an NH₄Cl-induced intracellular acid load (initial pH_i recovery rate 0.55±0.34 pH·min^-1) was nearly totally inhibited by 1 mmol·l^-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l^-1 bafilomycin A₁. In contrast, both vanadate (1 mmol·l^-1) and amiloride (1 mmol·l^-1) inhibited pH_i recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na⁺ from the bathing saline had no effect on pH_i recovery, indicating that a Na⁺/H⁺ exchange is not significantly involved in pH_i regulation. Instead pH_i regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO dimethylsulphoxide - DNP 2,4-dinitrophenol - NMDG N-methyl-D-glucamine - pH_i intracellular pH - pH_lu pH of the luminal fluid - pH_o pH of the superfusion medium - _I intrinsic intracellular buffer capacity     

Ion movements associated with chorion elevation during egg laying in trout (Oncorhynchus mykiss) in fresh water

Within 1 min of transfer from coelomic fluid to fresh water, eggs of rainbow trout (Oncorhynchus mykiss) underwent a transient loss of Na⁺ and K⁺ coupled with an elevation of the chorionic envelope. Both mechanisms were blocked by adding a monovalent cation Li⁺ or K⁺ (140 mmol·l^-1) to the fresh water, but the divalent ion Mg²⁺ (100 mmol MgCl₂·l^-1) or elevating the osmotic pressure to 300 mOsmol·l^-1 with glycine had no inhibitory effect. The blocking of Na⁺ loss occurred at external monovalent cation (LiCl) concentrations above 70 mmol·l^-1. A 20-s exposure of eggs to fresh water was sufficient to trigger Na⁺ loss and chorion elevation, even when the eggs were subsequently transferred to fresh water containing 140 mmol LiCl·l^-1. Eggs placed in a medium containing 140 mmol LiCl·l^-1 and 2 mmol Ca(NO₃)₂·l^-1 showed chorion elevation and associated Na⁺ loss after addition of calcium ionophore (20 mol·l^-1 A.23187). This activation by calcium ionophore was supressed in a Ca²⁺-free medium containing 5 mmol EGTA·l^-1.     

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     

Effect of extracellular calcium on the contractility of warm-and cold-acclimated crucian carp heart

Crucian carp (Carassius carassius L.) were acclimated for at least 4 weeks to 2°C or 22°C, and the consequences of thermal acclimation on force development, time-course of contraction and action potential duration of the ventricular myocardium were studied. In cold-acclimated fish contraction was activated at much lower external [Ca] than in warm-acclimated fish: [Ca] for half-maximal force was 0.9±0.15 and 3.1±0.92 mmol·l^-1 (P<0.05) for cold- and warm-acclimated fish, respectively. Durations of contraction and relaxation were significantly longer in fish acclimated to 2°C than in fish acclimated to 22°C, especially at [Ca] below 2 mmol·l^-1. In low-Ca solution ventricular action potential was prolonged both in cold- and warm-acclimated fish. In 0.5 mmol·l^-1 Ca action potential duration at zero voltage level was longer in cold- than warm-acclimated fish. Although lengthening of action potential was evident in both acclimation groups, a marked prolongation of contraction duration by low-Ca solutions occurred only in cold-acclimated fish. This suggests that a plateau component of contraction is present in cold-acclimated fish but less well developed in warm-acclimated fish hearts. Contractions were strongly inhibited by sarcolemmal Ca-channel blocker, cadmium (100 and 300 mol·l^-1), in both warm- and cold-acclimated crucian carp hearts. However, the sarcoplasmic reticulum Ca release channel blocker, ryanodine (10 mol·l^-1), had no effect on the force of contraction in either acclimation group. These results suggest that the contraction of crucian carp heart is controlled by sarcolemmal mechanisms without contribution by sarcoplasmic reticulum Ca release. Since the Ca sensitivity of myofilaments was not altered by thermal acclimation, the results indicate that thermal acclimation alters Ca activation of contraction of the crucian carp heart at the level of sarcolemma.Abbreviations AP action potential - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetra-acetic acid - F _max maximum force - F _max maximum rate of contraction - F _min maximum rate of relaxation - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - pCa log [Ca] - Pl action potential plateau - SL sarcolemma - SR sarcoplasmic reticulum - TPF time to peak force - T1/2R time to half relaxation from the peak force     

Metabolic correlates to glycerol biosynthesis in a freeze-avoiding insect,Epiblema scudderiana

Summary The course of glycerol biosynthesis, initiated by exposure to –4°C, was monitored in larvae of the goldenrod gall moth,Epiblema scudderiana, and accompanying changes in the levels of intermediates of glycolysis, adenylates, glycogen, glucose, fructose-2,6-bisphosphate, and fermentative end products were characterized. Production of cryoprotectant was initiated within 6 h after a switch from +16° to –4°C, with halfmaximal levels reached in 30 h and maximal content, 450–500 mol/g wet weight, achieved after 4 days. Changes in the levels of intermediates of the synthetic pathway within 2 h at –4°C indicated that the regulatory sites involved glycogen phosphorylase, phosphofructokinase, and glycerol-3-phosphatase. A rapid increase in fructose-2,6-bisphosphate, an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase, appeared to have a role in maintaining flux in the direction of glycerol biosynthesis. Analysis of metabolite changes as glycerol production slowed suggested that the inhibitory restriction of the regulatory enzymes was slightly out of phase. Inhibition at the glycerol-3-phosphatase locus apparently occurred first and resulted in a build-up of glycolytic intermediates and an overflow accumulation of glucose. Glucose inhibition of phosphorylase, stimulating the conversion of the activea to the inactiveb forms, appears to be the mechanism that shuts off phosphorylase function, counteracting the effects of low temperature that are the basis of the initial enzyme activation. Equivalent experiments carried out under a nitrogen gas atmosphere suggested that the metabolic make-up of the larvae in autumn is one that obligately routes carbohydrate flux through the hexose monophosphate shunt. The consequence of this is that fermentative ATP production during anoxia is linked to the accumulation of large amounts of glycerol as the only means of maintaining redox balance.Abbreviations G6P glucose-6-phosphate - F6P fructose-6-phosphate - F1, 6P fructose-1,6-bisphosphate - F2,6P ₂ fructose-2,6-bisphosphate - G3P grycerol-3-phosphate - DHAP dinydroxyacetonephosphate - GAP glyceraldehyde-3-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase - FBPase fructose-1,6-bisphosphatase - PK pyruvate kinase     

Solar heat gain in a desert rodent: unexpected increases with wind speed and implications for estimating the heat balance of free-living animals

We quantified metabolic power consumption as a function of wind speed in the presence and absence of simulated solar radiation in rock squirrels, Spermophilus variegatus, a diurnal rodent inhabiting arid regions of Mexico and the western United States. In the absence of solar radiation, metabolic rate increased 2.2-fold as wind speed increased from 0.25 to 4.0 m·s^-1. Whole-body thermal resistance declined 56% as wind speed increased over this range, indicating that body insulation in this species is much more sensitive to wind disruption than in other mammals. In the presence of 950 W·m^-2 simulated solar radiation, metabolic rate increased 2.3-fold as wind speed was elevated from 0.25 to 4.0 m·s^-1. Solar heat gain, calculated as the reduction in metabolic heat production associated with the addition of solar radiation, increased with wind speed from 1.26 mW·g^-1 at 0.25 m·s^-1 to 2.92 mW·g^-1 at 4.0 m·s^-1. This increase is opposite to theoretical expectations. Both the unexpected increase in solar heat gain at elevated wind speeds and the large-scale reduction of coat insulation suggests that assumptions often used in heat-transfer analyses of animals can produce important errors.Abbreviations absorptivity of coat to solar radiation - kinematic viscosity of air (mm²·s^-1) - reflectivity of coat to solar radiation - a r _B expected at zero wind speed (s·m^-1) - A _P projected surface area of animal on plane perpendicular to solar beam (cm²) - A _SKIN skin surface area (cm²) - b Coefficient describing change in r _B with change in square-root of wind speed (s^1.5·m^1.5) - d hair diameter (m) - d characteristic dimension of animal (m) - D _H thermal diffusivity of air (m²·s^-1) - E evaporative heat loss (W·m^-2) - I probability per unit coat depth that photon will strike hair - k constant equalling 1200 J·m^-3·°C^-1 - l _C coat depth m) - l _H hair length (m) - M metabolic rate (W·m^-2) - n density of hairs of skin (m^-2) - Q _A solar heat gain to animal (W·m^-2) - Q _I solar irradiance intercepted by animal (W·m^-2) - RQ respiratory quotient - r _A thermal resistance of boundary layer (s·m^-1) - r _B whole-body thermal resistance (s·m^-1) - r _E thermal resistance between animal surface and environment s·m^-1) - r _R radiative resistance (s·m^-1) - r _S sum of r _B and r _E at 0.25 m·s^-1 (s·m^-1) - r _T tissue thermal resistance s·m^-1) - T _AIR air temperature (°C) - T _B body temperature (°C) - T _E operative temperature of environment (°C) - T _ES standard operative temperature of environment (°C) - u wind speed (m·s^-1)     

Diving metabolism and thermoregulation in common and thick-billed murres

The diving and thermoregulatory metabolic rates of two species of diving seabrid, common (Uria aalge) and thick-billed murres (U. lomvia), were studied in the laboratory. Post-absorptive resting metabolic rates were similar in both species, averaging 7.8 W·kg^-1, and were not different in air or water (15–20°C). These values were 1.5–2 times higher than values predicted from published allometric equations. Feeding led to increases of 36 and 49%, diving caused increases of 82 and 140%, and preening led to increases of 107 and 196% above measured resting metabolic rates in common and thick-billed murres, respectively. Metabolic rates of both species increased linearly with decreasing water temperature; lower critical temperature was 15°C in common murres and 16°C in thick-billed murres. Conductance (assuming a constant body temperature) did not change with decreasing temperature, and was calculated at 3.59 W·m^-2·^oC^-1 and 4.68 W·m^-2·^oC^-1 in common and thick-billed murres, respectively. Murres spend a considerable amount of time in cold water which poses a significant thermal challenge to these relatively small seabirds. If thermal conductance does not change with decreasing water temperature, murres most likely rely upon increasing metabolism to maintain body temperature. The birds probably employ activities such as preening, diving, or food-induced thermogenesis to meet this challenge.Abbreviations ADL aerobic dive limit - BMR basal metabolic rate - FIT food-induced thermogenesis - MHP metabolic heat production - MR metabolic rate - PARR post-absorption resting rate - RMR resting metabolic rate - RQ respiratory quotient - SA surface area - STPD standard temperature and pressure (25°C, 1 ATM) - T _a ambient temperature - T _b body temperature - T _IC Iower critical temperatiure - TC thermal conductance - V oxygen consumption rate - W body mass     

The effect of temperature on the pattern of torpor in a marsupial hibernator

Physiological variables of torpor are strongly temperature dependent in placental hibernators. This study investigated how changes in air temperature affect the duration of torpor bouts, metabolic rate, body temperature and weight loss of the marsupial hibernator Burramys parvus (50 g) in comparison to a control group held at a constant air temperature of 2°C. The duration of torpor bouts was longest (14.0±1.0 days) and metabolic rate was lowest (0.033±0.001 ml O₂·g^-1·h^-1) at2°C. At higher air temperatures torpor bouts were significantly shorter and the metabolic rate was higher. When air temperature was reduced to 0°C, torpor bouts also shortened to 6.4±2.9 days, metabolic rate increased to about eight-fold the values at 2°C, and body temperature was maintained at the regulated minimum of 2.1±0.2°C. Because air temperature had such a strong effect on hibernation, and in particular energy expenditure, a change in climate would most likely increase winter mortality of this endangered species.Abbreviationst STP standard temperature and pressure - T _a air temperature - T _b body temperature - VO₂ rate of oxygen consumption     

Induction of a high-capacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism

Roots of nitrate-starved and nitrate-pretreated seedlings of Hordeum vulgare were used to investigate the induction of a high-capacity uptake mechanism for nitrate. When exposed to 0.2 mmol·l^-1KNO₃, nitrate-starved roots took up nitrate at a rate of approx. 1 mol·(g FW)^-1·h^-1; K⁺ was absorbed at a rate ten-times higher. Nitrate uptake accelerated after a lag of about 1 h, until it matched the rate of K⁺ uptake about 4 h later. p-Fluorophenylalanine (FPA), which prevents the synthesis of functioning proteins, suppressed the development of the high-capacity mechanism. Pretreatment of the roots with 0.2 mmol·l^-1 Ca(NO₃)₂ for 24 h established the high-capacity mechanism. Pretreated roots were able to absorb nitrate at high rates immediately upon exposure to 0.2 mmol·l^-1KNO₃, in the absence or presence of FPA. The high-capacity mechanism, once established, appeared to have a protein turnover as slow as that of the low-capacity mechanism or that of the mechanism involved in the uptake of K⁺. In contrast, the mechanisms for the transport of nitrate and K⁺ into the xylem vessels were completely blocked by FPA within 1 h of application, confirming earlier evidence for a rapid turnover of the transport proteins in the xylem parenchyma.Nitrate reduction proceeded at rates which were roughly one-tenth as large as the rates of the respective nitrate-uptake processes, indicating that nitrate-reductase activity was determined by the rate of nitrate uptake and not vice versa.We conclude that the formation of a high-capacity nitrate-uptake mechanism in barley roots occurs in response to nitrate uptake through a constitutive mechanism of low capacity which appears to function as a sensing mechanism for nitrate in the environment of the roots.Abbreviation FPA p-fluorophenylalanine