Photosynthesis and successional status of Costa Rican rain forest trees

Seven tree species from three different light environments in the wet lowland forests of Costa Rica were grown under controlled environment conditions to assess light related photosynthetic potentials. Light saturated photosynthesis rates were clearly related to light levels of the field environments. Mean saturated, net photosynthetic rates ranged from 6.8 to 11.3 to 27.7 mol m^–2 sec^–1 for plants from heavy shade, canopy light gaps and man-made clearings respectively. Light saturation of plants from clearings occurred at photosynthetic photon flux densities greater than 1000 mol m^–2 sec^–1 whereas plants from heavy shade environments became light saturated near 500 mol m^–2 sec^–1. Plants that normally occur in intermediate light environments were intermediate in light saturation levels. Mean maximum stomatal conductances ranged from 1.0 to 7.3 mm sec^–1 and followed a pattern similar to photosynthetic rates.     

pH equilibration in human erythrocyte suspensions

Summary A stopped-flow rapid reaction apparatus was used to study the rate of pH equilibration in human red cell suspensions. Flux of OH^– or H⁺ was determined over a wide range of extracellular pH (4–11) in CO₂-free erythrocyte suspensions. In these experiments, an erythrocyte suspension at pH 7.3 is rapidly mixed with an equal volume of NaCl solution at 3.0>pH>11.5. The pH of the extracellular fluid of the mixture changes rapidly as OH^– or H⁺ moves across the red cell membrane. Flux and velocity constants can be calculated from the initiald pH/dt using the known initial intra- and extracellular pH. It was found that the further the extracellular pH is from 7.3 (in either direction from 4–11), the greater the absolute value of total OH^– and/or H⁺ flux. Pretreatment with SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid), a potent anion exchange inhibitor, greatly reduces flux over the entire pH range, while exposure to valinomycin, a potassium ionophore, has no measurable effect. These data suggest that (i) both H⁺ and OH^– may be moving across the red cell membrane at all pH; (ii) the species dominating pH equilibration is probably dependent on the extracellular pH, which determines the magnitude of the driving gradient for each ion; and (iii) the rapid exchange pathway of the erythrocyte membrane may be utilized for both H⁺ and OH^– transport during CO₂-free pH equilibration.     

Proton conductance through phospholipid bilayers: Water wires or weak acids?

The proton/hydroxide (H⁺/OH^–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H⁺/OH^– transport are unknown. This review describes the characteristics of H⁺/OH^– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H⁺/OH^– conductances (G _H/OH) range from 2–6 nS cm^–2, corresponding to net H⁺/OH^– permeabilities of (0.4–1.7)×10^–5 cm sec^–1. Inhibitors ofG _H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG _H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG _H/OH are not correlated. The characteristics ofG _H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG _H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG _H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG _H/OH remains which may be due to water wires, hydrated defects, or other mechanisms.     

Extracellular amylolytic system of the yeast Lipomyces kononenkoae

Summary A strain of the yeast Lipomyces kononenkoae which converted starch into SCP with a high yield, produced three extracellular amylases which were purified from the culture fluid by Ficoll concentration, dialysis, isopropanol precipitation and DE-cellulose chromatography: an -amylase, a glucoamylase and a debranching transferase. The latter transferred -1,6-glucosyl units from panose to glucose forming maltose and appeared to have some debranching activity on amylopectin. The -amylase had the following properties: MW 38000 daltons; no effect of added calcium ions on activity; optimum temperature and pH for activity around 40°C and pH 5.5; H and S of heat inactivation 24360 cal mol^–1 and 29.2 cal deg^–1 mol^–1; range of pH stability pH 4–6.5; pI=7.1; final low molecular weight products of starch hydrolysis, maltose and glucose; K_m (40°C, pH 5.5) for starch 2.7 gl^–1, for maltotriose 109 gl^–1; uncompetitive inhibition by maltose with K_i (40°C, pH 5.5) 29.5 gl^–1. The glucoamylase had the following properties: MW 81500 daltons; optimum temperature and pH for activity around 50°C and pH 4.5: H and S of heat inactivation 20400 cal mol^–1 and 17.7 cal deg^–1 mol^–1; range of pH stability pH 4–6.5; pI=6.1; K_m (30°C, pH 4.5) for soluble starch 16.2 gl^–1, for maltose 0.36 gl^–1, for p-nitrophenyl--D-glucoside 0.35 gl^–1; competitive inhibition by glucose with K_i (30°C, pH 4.5) 4.7 gl^–1.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.     

pH Modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistiguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pH_int/pH_ext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2m). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pH_ext 5.8 (K _m =1.33vs. 1.70 m;V _max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect thatK _m, for carrier-mediated F transport. These data are consistent with similarK _i's for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H⁺ K _m (or OH^– IC₅₀) corresponding to pH 4.89. External pH affects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺) rather than competitive binding that is mutually exclusive.     

Effect of dichlorophenolindophenol, dichlorophenolindophenol-sulfonate, and cytochromec on redox capacity and simultaneous net H+/K+ fluxes in aeroponically grown seedling roots of sunflower (Helianthus annuus L.): New evidence for a plasma membrane CN−-resistant redox chain

Summary Excised roots from axenically grown sunflower seedlings reduced or oxidized exogenously added 2,6-dichlorophenolindophenol (DCIP), DCIP-sulfonate (DCIP-S), and cytochromec, and affected simultaneous H⁺/K⁺ net fluxes. Experiments were performed with nonpretreated living and CN^–-pretreated poisoned roots (control and CN^–-roots). CN^–-roots showed no H⁺/K⁺ net flux activity but still affected the redox state of the compounds tested. The hydrophobic electron acceptor DCIP decreased the rate of H⁺ efflux in control roots with extension of the maximum rate and optimal pH ranges, then the total net H⁺ efflux (H⁺) equalled that of the roots without DCIP. The simultaneously measured K⁺ influx rate was first inhibited, then inverted into efflux, and finally influx recovered to low rates. This effect could not be due to uptake of the negatively charged DCIP, but due to the lower H⁺ efflux and the transmembrane electron efflux caused by DCIP, which would depolarize the membrane and open outward K⁺ channels. The different H⁺ efflux kinetics characteristics, together with the small but significant DCIP reduction by CN^–-roots were taken as evidence that an alternative CN^–-resistant redox chain in the plasma membrane was involved in DCIP reduction. The hydrophilic electron acceptor DCIP-S enhanced both H⁺ and K⁺ flux rates by control roots. DCIP-S was not reduced, but slightly oxidized by control roots, after a lag, while CN^–-roots did not significantly oxidize or reduce DCIP-S. Perhaps the hydrophobic DCIP could have access to and drain electrons from an intermediate carrier deep inside the membrane, to which the hydrophilic DCIP-S could not penetrate. Also cytochromec enhanced H⁺ and K⁺, consistent with the involvement of the CN^–-resistant redox chain. Control roots did not reduce but oxidize cytochromec after a 15 min lag, and CN^–-roots doubled the rate of cytochromec oxidation without any lag. NADH in the medium spontaneously reduced cytochromec, but control or CN^–-roots oxidized cytochromec, despite of the presence of NADH. In this case CN^–-roots were less efficient, while control roots doubled the rate of cytochromec oxidation by CN^–-roots, after a 10 min lag in which cytochromec was reduced at the same rate as the medium plus NADH did. CN^–-roots seemed to have a fully activated CN^–-resistant branch. The described effects on K⁺ flux were consistent with the current hypothesis that redox compounds changed the electric membrane potential (de- or hyperpolarization), which induces the opening of voltage-gated in- or outward K⁺ channels.Abbreviations Cyt c cytochromec - DCIP 2,6-dichlorophenolindophenol - DCIP-S 2,6-dichlorophenolindophenol 3-sulfonate - HCF(III) hexacyanoferrate (III) - PM plasma membrane - SHAM salicylhydroxamic acid - VH⁺ and VK⁺ H⁺ efflux and K⁺ influx rates - H⁺ and K⁺ total H⁺ efflux and K⁺ influx at the end of the experiment - H⁺ and K⁺ buffering power of the titrated medium     

Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H⁺ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl^– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl^–=Br^–=I^–>SO ₄ ^2– F^–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl^–-flux measurements and studies on the H⁺ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H⁺ uptake into the endosomes.     

Taurine transport by rabbit kidney brush-border membranes: Coupling to sodium,chloride, and the membrane potential

Summary Ion dependence and electrogenicity of taurine uptake were studied in rabbit renal outer cortical brush-border membrane vesicles isolated by differential precipitation. Na⁺-d-glucose cotransport was followed in parallel to monitor changes in the membrane potential. Concentrative taurine flux was dependent on a chemical and/or an electrical Na⁺ gradient (K⁺ diffusion potential) and could be completely inhibited by other -amino acids. It displayed a specific anion requirement (Cl^–Br^–SCN^–>I^–>NO ₃ ^– ). At chemical Na⁺ equilibrium, Cl^– gradients, depending on their orientation, stimulated or inhibited taurine uptake more than could be attributed solely to electrical anion effects, although a Cl^– gradient alone could not energize an overshoot. Furthermore, taurine tracer exchange was significantly stimulated by Cl^– as well as Br^–. The Cl^– stoichiometry was found to be one, whereas taurine transport, in the presence of Cl^–, was sigmoidally related to the Na⁺ concentration, resulting in a coupling ratio of 2 to 3 Na⁺: 1 taurine. Upon Cl^– replacement with gluconate, taurine uptake showed a reduced potential sensitivity and was no longer detectably affected by the Na⁺ concentration (up to 150mm). These results suggest a 2 to 3 Na⁺:1 Cl^–:1 taurine cotransport mechanism driven mainly by the Na⁺ gradient, which is sensitive to the membrane potential due to a negatively charged empty carrier. Cl^– appears to stimulate taurine flux primarily by facilitating the formation of the translocated solute-carrier complex.     

Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H⁺/e^– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m^–2) (strong light) the H⁺/e^– ratio was 3. At low intensity (0.9 W · m^–2) (weak light) the H⁺/e^– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H⁺/e^– ratio due to underestimation of the initial rate of H⁺ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.     

Concentration and flux of solutes from snow and forest floor during snowmelt in the West-Central Adirondack region of New York

Decreases in pH and increases in the concentration of Al and NO ₃ ^– have been observed in surface waters draining acid-sensitive regions in the northeastern U.S. during spring snowmelt. To assess the source of this acidity, we evaluated solute concentrations in snowpack, and in meltwater collected from snow and forest floor lysimeters in the west-central Adirondack Mountains of New York during the spring snowmelt period, 29 March through 15 April 1984.During the initial phase of snowmelt, ions were preferentially leached from the snowpack resulting in elevated concentrations in snowmelt water (e.g. H⁺ = 140 eq.l^–1; NO ₄ ^2– = 123 eq.l^–1; SO ₃ ^– = 160 eq.l^–1). Solute concentrations decreased dramatically within a few days of the initial melt (< 50 eq.l^–1). The concentrations of SO ₄ ^2– and NO ₃ ^– in snowpack and snowmelt water were similar, whereas NO^– ₃ in the forest floor leachate was at least two times the concentration of SO ₄ ^2– .Study results suggest that the forest floor was a sink for snowmelt inputs of alkalinity, and a net source of H⁺, NO ₃ ^– , dissolved organic carbon, K⁺ and Al inputs to the mineral soil. The forest floor was relatively conservative with respect to snowmelt inputs of Ca²⁺, SO ₄ ^2– and Cl^–. These results indicate that mineralization of N, followed by nitrification in the forest floor may be an important process contributing to elevated concentrations of H⁺ and NO ₃ ^– in streams during the snowmelt period.     

Characterization of MgATP-Driven H+ uptake in to a microsomal vesicle franction from rat pancreatic acinar cells

Summary In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H⁺ transport was evaluated by measuring H⁺-dependent accumulation of acridine orange (AO). Active H⁺ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (K _m for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H⁺ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H⁺ accumulatioin was abolished by protonophores. such as nigericin (10^–6 mol/liter) or CCCP (10^–5 mol/liter), and by inhibitors of nonmitochondria H⁺ ATPase, such as NEM or NBD-Cl, at a concentration of 10^–5 mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H⁺ pumps, such as DCCD (10^–5 mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H⁺ transport by about 90%. Vanadate (2×10^–3 mol/liter). a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H⁺ transport. The stilbene derivative DIDS (10^–4 mol/liter), which inhibits anion transport systems, abolished H⁺ transport completely. MgATP-dependent H⁺ transport was found to be anion dependernt in the sequence Cl^–>Br^–>gluconate^–; in the presence of SO ₄ ^–2 . CH₃COO^– or No ₃ ^– , no H⁺ transport was observed. MgATP-dependent H⁺ accumulation was also cation dependent in the sequence K⁺>Li⁺>Na⁺=choline⁺, As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl^– and a K⁺ conductance, as well as a small H⁺ conductance. were found in the microsomal membranes. When membranes containing the H⁺ pump wer further purified by Percoll gradient centrifugatin (ninefold enrichment comparad to homogenate), no correlation with markers for endoplasmic reticulum., mitochondria, plasma membranes, zymogen graules or Golgi membranes was found.The present data indicate that the H⁺ pump located in microsomes from rat exocrine pancreas is a vacuolar-or V-type H⁺ ATPase and has most similarities to that described in endoplasmic reticulum. Golgi apparatus or endosomes.     

pH-stat experiments in proximal renal tubules

Summary The pH-stat technique has been used to measure H⁺ fluxes in gastric mucosa and urinary bladder in vitro while keeping mucosal pH constant. We now report application of this method in renal tubules. We perfused proximal tubules with double-barreled micropipettes, blocked luminal fluid columns with oil and used a double-barreled Sb/reference microelectrode to measure pH, and Sb or 1n HC1-filled microelectrodes to inject OH^– or H⁺ ions into the tubule lumen. By varying current injection, pH was kept constant at adjustable levels by an electronic clamping circuit. We could thus obtain ratios of current (nA) to pH change (apparent H⁺-ion conductance). These ratios were reduced after luminal 10^–4 m acetazolamide, during injection of OH^–, but they increased during injection of H⁺. The point-like injection source causes pH to fall off with distance from the injecting electrode tip even in oil-blocked segments. Therefore, a method analogous to cable analysis was used to obtain H⁺ fluxes per cm² epithelium. The relation betweenJ _H ⁺ and pH gradient showed saturation kinetics of H fluxes, both during OH^– and H⁺ injection. This kinetic behavior is compatible with inhibition ofJ _H by luminal H⁺. It is also compatible with dependence on Na⁺ and H⁺ gradients of a saturable Na/H exchanger. H⁺-ion back-flux into the tubule lumen also showed saturation kinetics. This suggests that H⁺ flow is mediated by a membrane component, most likely the Na⁺–H⁺ exchanger.     

The effects of pH and calcium concentrations on gill potentials in the Brown Trout,Salmo trutta

Summary Measurements of the transepithelial potential (V_int-V_ext) across the gills of Brown Trout,Salmo trutta, were made in solutions of a range of pH and calcium concentrations. The potential was strongly dependent on external pH, being negative in neutral solutions but positive in acid solutions. The addition of calcium to the external medium produced a positive shift in potential in all but very acid media (pH 4.0–3.5), where very little change was seen. The gill membrane appears to act as a hydrogen electrode having a very high permeability to H⁺ ions, and the potential behaves as a diffusion potential. The presence of calcium reduced the permeability to both H⁺ and Na⁺ ions but even at a calcium concentration of 8.0 mM/l the permeability ratio H⁺/Na⁺ was still more than 900. The transepithelial potential is shown to be diffusional in origin and is discussed in terms of the relative permeability of the gill to H⁺, Na⁺ and Cl^– ions. Sodium fluxes across the gills were measured and provide the basis for a theoretical consideration of Na⁺, Cl^– and H⁺ fluxes across the gills in neutral and acid solutions. The positive potential at low pH largely accounts for the increased loss of sodium from fish in these conditions.     

Effects of pH,potential, chloride and furosemide on passive Na+ and K+ effluxes from human red blood cells

Summary Ouabain-resistant effluxes from pretreated cells containing K⁺/Na⁺=1.5 into K⁺ and Na⁺ free media were measured.Furosemide-sensitive cation effluxes from cells with nearly normal membrane potential and pH were lower in NO ₃ ^– media than in Cl^– media; they were reduced when pH was lowered in Cl^– media. When the membrane potential was positive inside furosemide increased the effluxes of Na⁺ and K⁺ (7 experiments). With inside-positive membrane potential thefurosemideinsensitive effluxes were markedly increased, they decreased with decreasing pH at constant internal Cl^– and also when internal Cl^– was reduced at constant pH. The correlation between cation flux and the membrane potential was different for cells with high or low internal chloride concentrations. The data with chloride47mm showed a better fit with the single-barrier model than with the infinite number-of-barriers model. With low chloride no significant correlation between flux and membrane potential was found. The data are not compatible with pure independent diffusion of Na⁺ and K⁺ in the presence of ouabain and furosemide.     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: Dependence on medium osmolality and regulation by signalling systems

Summary To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and ⁸⁶Rb and ²²Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 l per 10⁶ cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P< 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by 7%, P<0.05) and by ethylisopropyl amiloride (by 13%, P< 0.005). Ouabain-sensitive ⁸⁶Rb influx was decreased by 30–40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an eightfold increase in bumetanide-inhibited ⁸⁶Rb influx and ⁸⁶Rb efflux. The (ouabain and bumetanide)-insensitive component of ⁸⁶Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive ⁸⁶Rb efflux was increased by 1.5–2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited ²²Na influx was increased by sixfold following osmotic-shrinkage of VSMC. The data show that both Na⁺/H⁺ exchange and Na⁺/K⁺/2Cl^– cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na⁺/K⁺/2Cl^– cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na⁺/K⁺/2Cl^– cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na⁺/H⁺ exchange activity. NaF increased Na⁺/H⁺ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na⁺/K⁺/2Cl^– cotransporter and the Na⁺/H⁺ exchanger.The authors gratefully acknowledge the financial support of the Swiss National Foundation, grant No. 3.817.087. Bernadette Weber is thanked for preparing the figures.     

Photosynthetic production of hydrogen peroxide by Ulva rigida C. Ag. (Chlorophyta)

Production of hydrogen peroxide has been found in Ulva rigida (Chlorophyta). The formation of H₂O₂ was light dependent with a production of 1.2 mol·g FW^–1·h^–1 in sea water (pH 8.2) at an irradiance of 700 mol photons m^–2·s^–1. The excretion was also pH dependent: in pH 6.5 the production was not detectable (< 5 nmol·g FW^–1·h^–1) but at pH 9.0 the production was 5.0 mol·g FW^–1·h^–1. The production of H₂O₂ was totally inhibited by 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). The ability of U. rigida growing in tanks (7501) under a natural light regime to excrete H₂O₂ was checked and found to be seven times higher at 08.00 hours than other times of the day. The H₂O₂ concentration in the cultivation tank (density: 2 g FW·l^–1) reached the highest value (3 M) at 11.00 hours. Photosynthesis was not influenced by H₂O₂ formation. The H₂O₂ is suggested to come from the Mehler reaction (pseudocyclic photophosphorylation). With an oxygen evolution of 120 mmol·g FW^–1·h^–1 at pH 8.2 and 90 mmol·g FW^–1·h^–1 at pH 9.0, 0.5% and 2.7% of the electrons were used for extracellular H₂O₂ production. The H₂O₂ production is sufficiently high to be of physiological and ecological significance, and is suggested to be a part of the defence against epi and endophytes.Abbreviations ACL artificial, continuous light - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - GNL greenhouse - LDC Luminol-dependent chemiluminescence - SOD Superoxide dismutase This investigation was supported by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Hierta-Retzius Foundation, Marianne and Marcus Wallenberg Foundation, the Swedish Environmental Protection Board, and CICYT Spain.     

Elektron-Spin-Resonanz-Untersuchungen der Reaktionskinetik strahleninduzierter freier Radikale des Cholesterins

Zusammenfassung Die Reaktionskinetik strahleninduzierter freier Radikale des Cholesterins wurde in flüssiger Phase bei Raumtemperatur mittels ESR-Spektroskopie untersucht. Mit Hilfe eines geeigneten photochemischen Initiationssystems ließen sich in Cyclohexanlösung unter UV-Bestrahlung (235 nm265 nm) genau dieselben freien Radikale des Cholesterins darstellen, die schon früher [9, 7] in röntgenbestrahltem Cholesterinpulver beobachtet worden waren. Bei ausreichendem O₂-Partialdruck (3·10⁴Torr) über der Probenlösung trat das ESR-Spektrum eines Peroxyradikals auf, das mittels der Analyse seiner Reaktionsprodukte (7-Hydroxy-Cholesterin und 7-Keto-Cholesterin) mit dem Cholesteryl-7-peroxyradikal identifiziert wurde. Die Kinetik sowohl der Bildung als auch des Zerfalls des Radikals entsprachen einer Reaktion von 2. Ordnung. Die Geschwindigkeitskonstante für den bimolekularen Zerfall, eine Disproportionierung in Alkohol und Keton unter Abgabe eines Moleküls O₂, wurde bei Raumtemperatur zuk ₂=(1,8 _–0,6 ^+0,9 )·10⁶ sec^–1M^–1·l bestimmt. Ferner wurde gezeigt, daß das Cholesteryl-7-peroxyradikal aus dem freien Radikal Cholesteryl-7 durch Anlagerung eines Moleküls O₂ entsteht. Für die Geschwindigkeitskonstante dieser Reaktion ergab sich eine untere Schranke vonk ₁=0,40·10¹⁰ sec^–1M^–1·l.

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Electron spin resonance investigations on radiation-induced free radicals of cholesterol in liquid phase

Summary The reaction kinetics of radiation-induced free radicals of cholesterol was studied in liquid phase at room temperature by means of e.s.r. spectroscopy on a solution of cholesterol in cyclohexane. Using a convenient photochemical initiation system, just those free radicals of cholesterol could be generated by the filtered u.v. radiation from a Xe high pressure lamp (235 nm265 nm) as were observed already a decade ago by Gordy [9] and by Ehrenberg, Löfroth [7] in X-irradiated cholesterol powder. At sufficiently high O₂-pressures (3·10^–4 Torr) over the sample solution a peroxy radical e.s.r. spectrum arose during u.v. irradiation which was identified by product analysis (7-hydroxy-cholesterol and 7-keto-cholesterol) to be dueto a cholesteryl-7-peroxyradical. The radical'sgeneration and decay kinetics was governed by a second order reaction. The velocity constant for bimolecular decay of the cholesteryl-7-peroxyradical was found to be k₂=(1.8 _–0,6 ^+0,9 )·10⁶sec^–1M^–1·l at room temperature. Furthermore it could be shown that the cholesteryl-7-peroxyradical was built up by the addition of one molecule of O₂ to a cholesteryl-7 free radical. For this reaction a value ofk ₁=0.4·10¹⁰ sec^–1 M^–1·l was estimated as a lower limit of the velocity constant.

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Die Arbeit stellt einen Auszug aus einer Dissertation an der Technischen Hochschule München dar.