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     

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     

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     

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     

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     

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.     

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     

A study on Na+-coupled oxidative phosphorylation: ATP formation supported by artificially imposed ΔpNa and ΔpK inVibrio alginolyticus cells

Addition of Na⁺ to the K⁺-loadedVibrio alginolyticus cells, creating a 250-fold Na⁺ gradient, is shown to induce a transient increase in the intracellular ATP concentration, which is abolished by the Na⁺/H⁺ antiporter, monensin. The pNa-supported ATP synthesis requires an additional driving force supplied by endogenous respiration or, alternatively, by a K⁺ gradient (high [K⁺] inside). In the former case, ATP formation is resistant to the protonophorous uncoupler. Dicyclohexylcarbodiimide and diethylstilbestrol, but not vanadate, completely inhibit Na⁺ pulse-induced ATP formation. The data agree with the assumption that Na⁺-ATP-synthase is involved in oxidative phosphorylation inV. alginolyticus. Interrelation of H⁺ and Na⁺ cycles in bacteria is discussed.Abbreviations and electrochemical gradients of H⁺ and Na⁺, respectively - transmembrane electric potential difference - pH, pNa, and pK concentration gradients of H⁺, Na⁺, and K⁺, respectively - CCCP carbonyl cyanidem-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diesthylstilbestrol - HQNO 2-heptyl-4-hydroxyquinolineN-oxide - Tricine N[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Catecholamine- and volume-dependent ion fluxes in carp (Cyprinus carpio) red blood cells

In carp erythrocytes, noradrenaline (10^-6 mol·l^-1) induces a 30- to 40-fold activation of Na⁺/H⁺ exchange (the ethylisopropylamiloride-inhibited component of the ²²Na influx) and a fourfold stimulation of the Na⁺, K⁺ pump (ouabain-inhibited component of ⁸⁶Rb influx). In both cases the effect of noradrenaline is blocked by propranolol but not phentolamine and is imitated by forskolin. An activator of protein kinase C (-phorbol 12-myristate, 13-acetate) increases Na⁺/H⁺ exchange by 10 times and decreases the Na⁺, K⁺ pump activity by 20–30 percent. In the presence of ethylisopropylamiloride the increment of the Na⁺, K⁺ pump activity induced by noradrenaline is reduced by 35–45 percent, indicating the existence of a Na⁺/H⁺ exchange-independent mechanism of the Na⁺, K⁺ pump regulation by -adrenergic catecholamines. Hypertonic shrinkage of carp erythrocytes results in a 40- to 80-fold activation of Na⁺/H⁺ exchange, whereas hypotonic swelling induces an increase in the rate of ⁸⁶Rb⁺ efflux which is inhibited by furosemide by about 30–40 percent. The rate of pH₀ recovery in response to acidification or alkalinization in rat erythrocytes is approximately 15 times as fast as in carp erythrocytes. Unlike in rat erythrocytes, valinomycin does not cause an alkalinization of incubation medium in carp erythrocytes indicating the absence of conductive pathway in the operation of anion transporter protein. A scheme is suggested which describes the interrelation of Na⁺/H⁺ exchange, Na⁺, K⁺ pump and a non-identified system providing for K⁺ efflux in cell swelling, regulation of cell volume and cytoplasmic pH in fish erythrocytes under conditions of deep hypoxia and high activity.Abbreviations cAMP cyclic adenosine monophosphate - CCCP carbonylcyamide m-chlorophenylhydrazone - DMSO dimethylsulphoxide - EIPA ethylisopropylamiloride - NA noradrenaline - PMA -phorbol 12-myristate, 13-acetate - RVD regulatory volume decrease - RVI regulatory volume increase     

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     

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     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

Na+ and K+ fluxes stimulated by Na+-coupled glucose transport: Evidence for a Ba2+-insensitive K+ efflux pathway in rabbit proximal tubules

Summary Addition of glucose or the nonmetabolizable analogue -methyl-d-glucoside to rabbit proximal tubules suspended in a glucoseand alanine-free buffer caused a sustained increase in intracellular Na⁺ content (+43±7 nmol · (mg protein)^–1) and a concomitant but larger decrease in K⁺ content (–72±11 nmol· (mg protein)^–1). A component of the net K⁺ efflux was Ba²⁺ insensitive, and was inhibited by high (1mm) but not low (10 m) concentrations of the diuretics, furosemide and bumetanide. The increase in intracellular Na⁺ content is consistent with the view that the increased rates of Na⁺ and water transport seen in the proximal tubule in the presence of glucose can be attributed (at least in part) to a stimulation of basolateral pump activity by an increased [Na⁺] _i .     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.     

Development of aldosterone-stimulation of short-circuit current across larval frog skin

Summary The short-circuit current (SCC) across isolated skin from bullfrog larvae in developmental stage XXI was small and insensitive to amiloride. Overnight incubation of this tissue with 10^-6 M aldosterone stimulated the SCC from 1.35±0.55 to 14.55±4.12 A·cm^-2 with 11.18±4.46 A·cm^-2 being blocked by 100 M amiloride. Histologic examination of aldosterone-treated skins revealed a separation of the apical cell layer from the underlying epidermis that was not seen in untreated preparations. The onset of amiloride-sensitive Na⁺ transport thus coincided with the exposure of the apical surface of newly differentiated epithelial cells. Similar results were obtained with skin from stage XXI larvae whose rate of metamorphosis had been stimulated by 10 g·l^-1 thyroxine (T₄) but not with skin from T₄-treated larvae in stages XIX and XX. Fluctuation analysis of the amiloride-sensitive SCC of the above preparations failed to show a consistent Lorentzian component in the power-density spectrum. Fluctuation analysis was possible on skins from larvae whose development had been accelerated by 7–9 days treatment with 10 g·l^-1 triiodothyronine (T₃). Aldosterone treatment of these tissues resulted in a significant increase in Na⁺ channel density.Abbreviations ASCC component of the short-circuit current (A·cm^-2) that is blocked by amiloride - fc frequency (Hz) at which the magnitude of the Lorenzian component of the power spectra is reduced by half - i current (pA) through individual amiloride-sensitive Na⁺ channels - I _Na+ amiloride-sensitive short-circuit current (A·cm^-2) that remains after treatment with a given amiloride concentration - k ₀₁ the rate constant (s^-1·M^-1) for the association of amiloride with Na⁺ channels - k ₁₀ rate constant (s^-1) for the dissociation of amiloride from Na⁺ channels - K _b magnitude of the power spectrum (A²·s·cm^-2) at a frequency of 1 Hz - KSCC short-circuit (A·cm^-2) current with K⁺ as the primary mucosal cation - M density of amiloride-sensitive Na⁺ channels in the apical cell membrane - SCC short-circuit current (A·cm^-2) - S _(f) magnitude of the power spectra (A²·s·cm^-2) at a given frequency - S ₀ the magnitude of the plateau region of the Lorentzian component of the power spectra (A²·s·cm^-2) - T ₃ Triiodothyronine - T ₄ Thyroxine     

Alanine and taurine transport by the gill epithelium of a marine bivalve: Effect of sodium on influx

Summary Marine mussels can accumulate amino acids from seawater into the epithelial cells of the gill against chemical gradients in excess of 5×10⁶ to 1. Uptake of both alanine and taurine into gill tissue isolated fromMytilus californianus was found to be dependent upon Na⁺ in the external solution. Uptake of these amino acids was described by Michaelis-Menten kinetics, and a reduction in external [Na⁺] (from 425 to 213mm) increased the apparent Michaelis constants (alanine, from 8 to 17 m; taurine, from 4 to 39 m) without a significant influence on theJ _max's of these processes. Fivemm harmaline, an inhibitor of Na-cotransport processes in many systems, reduced both alanine and taurine uptake by more than 95%; this inhibition appeared to be competitive in nature, with an apparentK _i of 43 m for the interaction with alanine uptake. Increasing the external [Na⁺] from 0 to 510mm produced a sigmoid activation of alanine and taurine uptake withK _Na's of approximately 325mm. The apparent Hill coefficients for this activation were 7.3 and 7.4 for alanine and taurine, respectively. These data are consistent with uptake mechanisms which require comparatively high concentrations of Na⁺ to activate transport, and which couple several Na⁺ ions to the transport of each amino acid. These characteristics, in conjunction with the previously demonstrated low passive permeability of the apical membrane to amino acids, result in systems capable of i) accumulating amino acids from seawater to help meet the nutritional needs of this animal, and ii) maintaining the high intracellular amino-acid concentrations associated with volume regulation in the gill.     

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     

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     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane