Substance P inhibits bicarbonate secretion from guinea pig pancreatic ducts by modulating an anion exchanger

The stimulatory pathways controlling HCO₃^- secretion by the pancreatic ductal epithelium are well described. However, only a few data are available concerning inhibitory mechanisms, which may play an important role in the physiological control of the pancreas. The aim of this study was to investigate the cellular mechanism by which substance P (SP) inhibits pancreatic ductal HCO₃^- secretion. Small intra/interlobular ducts were isolated from the pancreas of guinea pigs. During overnight culture the ducts seal to form a closed sac. Transmembrane HCO₃^- fluxes were calculated from changes in intracellular pH (measured using the pH-sensitive dye BCECF) and the buffering capacity of the cells. We found that secretin can stimulate HCO₃^- secretion in guinea pig pancreatic ducts about fivefold and that this effect could be totally blocked by SP. The inhibitory effect of SP was relieved by spantide, an SP receptor antagonist. SP had no effect on the activity of basolateral Na⁺-HCO₃^- cotransporters and Na⁺/H⁺ exchangers. However, the peptide did inhibit a Cl^--dependent HCO₃^- efflux (secretory) mechanism, most probably the Cl^-/HCO₃ exchanger on the apical membrane of the duct cell. pancreas; Cl^-/HCO₃^- exchanger; tachykinin     

Fatty acid synthesis by spinach chloroplasts I. Property of fatty acid synthesis from acetate

Intact chloroplasts (about 70% Class I chloroplasts) isolatedfrom spinach leaves incorporated 150 nmoles of [1-¹⁴C] acetateinto fatty acids per mg chlorophyll in 1 hr at pH 8.3, 25°Cand 25,000 lux. On electron and phase-contrast microscopiescombined with hypotonic treatment of chloroplasts, this syntheticactivity was shown to be proportional to the percentage of ClassI chloroplasts in the preparation. Light was necessary for thesynthesis, the activity in the complete reaction mixture inthe dark being only 2% of that in the light. The synthetic activityincreased with increasing intensities of light to reach saturationat 6,000 lux. CoA and ATP were most effective as cofactors,HCO₃^–, HPO₄^2–, Mg^2$ and Mn^2$ were less effective.ATP could be replaced by ADP in the presence of Pi, suggestingpossible supply of ATP by photophosphorylation. Omission ofthe NADPH-generation system and NADH did not affect the synthesis,indicating sufficient provision of endogenous NADPH and NADHin intact chloroplasts under light. Addition of DTE did notcause recovery of the synthetic activity of intact chloroplastsin the dark. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Coupling Ratios H+/e=3 versus H+/e=2 in Chloroplasts and Quantum Requirements of Net Oxygen Exchange during the Reduction of Nitrite, Ferricyanide or Methylviologen

Using intact and osmotically ruptured chloroplasts, ratios ofcoupling between deposition of protons in the intrathylakoidspace and light-dependent transport of electrons from waterto an external acceptor were determined. The data indicate couplingbetween proton and electron transport at a ratio of H⁺/e=3 withmethylviologen as electron acceptor in thylakoids and with nitriteas electron acceptor in intact chloroplasts. With ferricyanideas electron acceptor in thylakoids, values close to H⁺/e=2 wereobserved. Evidence is discussed that H⁺/e=3 is a fixed valuein intact chloroplasts at levels of thylakoid energization sufficientfor supporting effective carbon assimilation. In the presence of methylviologen and ascorbate, the minimumquantum requirement of oxygen uptake by thylakoids was about2.7 quanta of 675 nm light per O₂ indicating an e/O₂ ratio of1.33. In the absence of ascorbate, and with KCN present in additionto methylviologen, e/O₂ ratios up to 4 were observed. The minimumquantum requirement of oxygen evolution by thylakoids in thepresence of ferricyanide and by intact chloroplasts in the presenceof nitrite was about 8 quanta/O₂. (Received May 1, 1995; Accepted October 2, 1995)     

Effects of chloride ion on HILL reactions in Euglena chloroplasts

Effects of Cl^– and other anions on the rate of HILL reactionin Euglena chloroplasts were investigated. Cl^– acceleratedthe reaction rate with ferricyanide as HILL oxidant; Br^–,F^– and I^– were also effective; NO₃^–, PO₄^2–and SO₄^2– were less effective. Divalent cations, Ca²⁺and Mg²⁺, were also highly effective. The promoting effectsof these ions were highly dependent on pH and the nature andconcentration of the HILL oxidant used. Accelerating effectsof the ion increased with decreasing concentrations of ferricyanide.Generally, the stimulating effect of Cl^– was much moremarked at pH 7–7.5, with little effect at pH 5. Thus,the pH-activity relationship in the HILL reaction is more orless markedly modified by addition of ions. Cl^–, and other anions, accelerated the reaction by affectingonly the dark rate-limiting portion of the HILL reaction; thelight reaction constant remained uninfluenced. We inferred thatsome reaction step, at which ferricyanide receives electronfrom photosystem 2, is accelerated by Cl^– and other ions.Cl^– effects were rather small, or undetectable, with DPIPor p-benzoquinone as oxidants. (Received January 8, 1970; )     

The Mechanism of Photosynthetic Use of Bicarbonate by Hydrodictyon africanum

Hydrodictyon africanum can photosynthesize at high pH underconditions in which HCO₃^– rather than CO₂ is the carbonspecies entering the cell. A passive entry of HCO₃^– seemsunlikely; a metabolic HCO₃^– pump is proposed. It is possiblethat such a pump is related to a light-dependent reaction specificto the use of HCO₃^–. This reaction is dependent on photosystem2, but appears to be independent of ATP. These characteristicsare similar to those of active lightdependent Cl influx in H.africanum, and suggest a similar energy source for the two pumps.The HCO₃^– pump may be electrogenic.     

The Location of the Transporting System for Inorganic Carbon and the Nature of the Form Translocated in Chlamydomonas reinhardtii

The permeability of the plasmalemma of Chlamydomonas reinhardtiicells was increased by treatment with poly-L-lysine or dimethylsulphoxideas indicated by 3-phosphoglyceric acid dependent O₂ evolution.These treatments decreased the ability of the cells to accumulateinorganic carbon internally and hence their photosynthetic affinityfor inorganic carbon in the medium. With saturating light andinorganic carbon, the photosynthetic rate was less affectedby the poly-L-lysine and dimethylsulphoxide treatments. Thusthe poly-L-lysine and dimethylsulphoxide did not alter the activityof the chloroplasts but rather made the intracellular inorganiccarbon pool more freely exchangeable with the medium. It isconcluded that the transporting system for inorganic carbonis located at the plasmalemma. Treatment with Diamox, an inhibitor of carbonic anhydrase, didnot affect photosynthetic rate and accumulation of inorganiccarbon when CO₂ was supplied but strongly inhibited both parameterswhen HCO^–₃ was supplied. In a mutant of Chlamydomonasreinhardtii lacking a cell wall, carbonic anhydrase leaks tothe medium and uptake of inorganic carbon is much faster whenCO₂ is supplied than when HCO^–₃ is supplied. These resultssuggest that CO₂ rather than HCO^–₃ is the inorganic carbonspecies that is actively translocated across the plasmalemma. Key words: Chlamydomonas, Inorganic carbon uptake     

The Influence of NO-3 and NH+4 Nutrition on the Gas Exchange Characteristics of the Roots of Wheat (Triticum aestivum) and Maize (Zea mays) Plants

Respiratory oxygen consumption by roots was 1·4- and1·6-fold larger in NH⁺₄-fed than in NO^-₃-fed wheat (Triticumaestivum L.) and maize (Zea mays L.) plants respectively. Higherroot oxygen consumption in NH⁺₄-fed plants than in NO^-₃-fedplants was associated with higher total nitrogen contents inNH⁺4-fed plants. Root oxygen consumption was, however, not correlatedwith growth rates or shoot:root ratios. Carbon dioxide releasewas 1·4- and 1·2-fold larger in NO⁺3-fed thanin NH⁺₄-fed wheat and maize plants respectively. Differencesin oxygen and carbon dioxide gas exchange rates resulted inthe gas exchange quotients of NH^-₄-fed plants (wheat, 0·5;maize, 0·6) being greatly reduced compared with thoseof NO^-₃-fed plants (wheat, 1·0; maize, 1·1). Measuredrates of HCO^-₃ assimilation by PEPc in roots were considerablylarger in 4 mM NH⁺₄-fed than in 4 NO^-₃ plants (wheat, 2·6-fold;maize, 8·3-fold). These differences were, however, insufficientto account for the observed differences in root carbon dioxideflux and it is probable that HCO^-₃ uptake is also importantin determining carbon dioxide fluxes. Thus reduced root extension in NH⁺₄-fed compared with NO^-₃-fedwheat plants could not be ascribed to differences in carbondioxide losses from roots.Copyright 1993, 1999 Academic Press Triticum aestivum, wheat, Zea mays, maize assimilation, ammonium assimilation, root respiration     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport     

Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA

Human NBC3 is an electroneutral Na⁺/HCO₃^– cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO₃^– metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO₂ + H₂O HCO₃^– + H⁺ in many tissues. We investigated whether NBC3, like some Cl^–/HCO₃^– exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K_d = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 ± 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 µM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH_i) recovery rate by 31 ± 3, or 38 ± 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH_i recovery rate by 69 ± 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [-³²P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO₃^– transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct. pH regulation; bicarbonate transport; metabolon     

Transport and Fixation of Inorganic Carbon during Photosynthesis in Cells of Anabaena Grown under Ordinary Air: III. Some Characteristics of the HCO3--Transport System in Cells Grown under Ordinary Air

In cells of cyanobacterium Anabaena variabilis grown under ordinaryair (low-CO₂ cells), the transport of both CO₂ and HCO₃^–was significantly enhanced by Na⁺. This effect was pronouncedas the external pH increased. When low-CO₂ cells were treatedwith an inhibitor of carbonic anhydrase (CA), only CO₂ transportbut not HCO₃^– transport, was inhibited. The initial rateof photosynthetic carbon fixation as a function of the concentrationof internal inorganic carbon (IC) was practically the same irrespectiveof whether CO₂ or HCO₃^– was externally supplied. Theseresults suggest that IC is actively transported through theplasma membrane in a form of HCO₃^– probably by some transporterand that the transmembrane Na⁺ gradient is involved in thisIC transport system. Free CO₂ may be hydrated by CA to HCO₃^–and then transported to the cells by this transporter. On the other hand, CO₂ is actively taken up by cells grown withair containing 5% CO₂ (high-CO₂ cells) though the enhancingeffect of Na⁺ was much smaller in high- CO₂ cells than in low-CO₂cells. The initial rate of fixation as a function of internal IC concentrationindicated that the rate of the carboxylation reaction of accumulatedIC is higher in I0W-CO₂ cells than in high-CO₂ cells. The studieswith ethoxyzolamide indicated that even in low-CO₂ cells, CAdoes not function inside Anabaena cells. These results suggestthat inside the low-CO₂ cells of Anabaena, some mediator(s)facilitates the transport of IC to RuBPCase. (Received January 23, 1987; Accepted April 24, 1987)     

Effect of salts on the Hill reaction in sonicated spinach chloroplasts

KC1 stimulated the ferricyanide-Hill reaction at pH higher than6.0 and at ferricyanide concentrations lower than 1 mM in sonicatedspinach chloroplasts. However, KCl did not affect on the DCIP-Hillreaction at all. We concluded that Cl^- is not an essential factor,but only a stimulator of the oxygen-evolving reaction of chloroplasts. (Received March 7, 1974; )     

Three distinct mechanisms of HCO3- secretion in rat distal colon

HCO₃^– secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO₃^– secretion have revealed evidence of lumen Cl^– dependence, suggesting a role for apical membrane Cl^–/HCO₃^– exchange, direct examination of HCO₃^– secretion in isolated crypt from rat distal colon did not identify Cl^–-dependent HCO₃^– secretion but did reveal cAMP-induced, Cl^–-independent HCO₃^– secretion. Studies were therefore initiated to determine the characteristics of HCO₃^– secretion in isolated colonic mucosa to identify HCO₃^– secretion in both surface and crypt cells. HCO₃^– secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO₃^– secretion (5.6 ± 0.03 µeq·h^–1·cm^–2) was abolished by removal of either lumen Cl^– or bath HCO₃^–; this Cl^–-dependent HCO₃^– secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h^–1·cm^–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl^– channel blocker. 8-Bromo-cAMP induced Cl^–-independent HCO₃^– secretion (and also inhibited Cl^–-dependent HCO₃^– secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl^–-independent, DIDS-insensitive, saturable HCO₃^– secretion that was not inhibited by NPPB. Three distinct HCO₃^– secretory mechanisms were identified: 1) Cl^–-dependent secretion associated with apical membrane Cl^–/HCO₃^– exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO₃^– exchange. chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat     

Vieillissement de l'appareil photosynth?tique II. Effet synergique de la lumi?re et du vieillissement in vitro sur les activit?es photochimiques de chloroplastes isol?s d'?pinard

The consequences of chloroplast ageing in vitro were furtherinvestigated, especially on the photochemical activities ofthese organelles. Ageing of chloroplasts in dark was accompanied by decreasesin activities for photohydrolysis and cyclic and non-cyclicsyntheses of ATP, photoreduction of NADP⁺ and O₂ evolution;but there was no decrease in ferricyanide photoreduction. Therates of decrease in these activities were comparable to therate of increase in chloroplast volume. Complete inhibitionswere reached when maximum chloroplast swelling had occurred,i.e. after 5 to 6 hr of incubation at 20?C in a Tris-NaCl (pH8) medium. Ageing in the light resulted in much accelerateddecreases in activities tested; the loss of capacity for light-inducedshrinkage was also accelerated by the light during ageing. Thus,light acts synergetically towards the ageing process. Moreover,light and, to a less extent, dark ageing, resulted in an uncouplingof chloroplast photophosphorylation and associated electronflow measured by ferricyanide photoreduction. The part of theelectron flow which is induced by coupling (+ ADP, Pi, MgCl₂,pH 8) or by uncoupling (+ NH₄C1, pH 7) was found to be verysensitive to light ageing. The NADP⁺ photoreduction loss wasrestored by addition of the ascorbate-DCPIP electron donor system,suggesting that ageing interferes with the integrity of photosystemII. In many respects, these effects of ageing are comparable withthe action of detergents and fatty acids on the structure andphotochemical activities of chloroplasts, especially in thatthey attack the energy transducing mechanism in chloroplasts. (Received May 24, 1969; )     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

Nongenomic regulation by aldosterone of the epithelial NHE3 Na(+)/H(+) exchanger

The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO₃^– absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here, we examined the transport mechanism(s) responsible for this regulation, focusing on Na⁺/H⁺ exchangers (NHE). In the MTAL, apical NHE3 mediates H⁺ secretion necessary for HCO₃^– absorption; basolateral NHE1 influences HCO₃^– absorption by regulating apical NHE3 activity. In microperfused rat MTALs, the addition of 1 nM aldosterone rapidly decreased HCO₃^– absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na⁺/H⁺ exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na⁺/H⁺ exchange activity over the intracellular pH range from 6.5 to 7.7, due to a decrease in V_max. Inhibition of HCO₃^– absorption by aldosterone was not affected by 0.1 mM lumen Zn²⁺ or 1 mM lumen DIDS, arguing against the involvement of an apical H⁺ conductance or apical K⁺-HCO₃^– cotransport. These results demonstrate that aldosterone inhibits HCO₃^– absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger. thick ascending limb; acid-base transport; epithelial Na⁺ transport; kidney     

Analogue Inhibition of the Active HCO-3 Transport Site in the Characean Plasma Membrane

Competitive inhibition of the HCO^–₃ transport site, atthe plasmalemma of Chara coraUina, by the CO^2–₃ ion isdemonstrated. This CO^2–₃ inhibition was used to demonstratethat HCO^–₃ ions enter the cell by facilitated ‘diffusion’when the HCO^–₃ transport system has been inactivated bytreatment with 10 mM K⁺. Use of CO^2–₃ as a HCO^–₃analogue is limited, however, because of the necessity to employsolutions of high pH. Inhibition was not observed in the presenceof a range of organic and inorganic acid anions. These resultsdemonstrate the stereo-specific nature of the HCO^–₃ bindingsite. A variety of amino compounds were found to inhibit H¹⁴CO^–₃influx. Inhibition appeared to be competitive, being completelyrelieved at higher substrate (HCO^–₃) concentrations. Asimple correlation was not found between the degree of inhibitionand the concentration of neutral base. A combination of thepresence of neutral base and experimental pH values of at least8·0 was required to produce the reactive species thatinhibited HCO^–₃ transport. This species is consideredto be the amino carbamate. These results are discussed withrespect to further HCO^–₃ analogue experiments.     

Photoproduction and Detoxification of Hydroxyl radicals in Chloroplasts and Leaves and Relation to Photoinactivation of Photosystems I and II

The light-dependent production of hydroxyl radicals (HO{dot})by thylakoids, chloroplasts and leaves of Spinacia oleraceawas investigated using dimethylsulfoxide as HO{dot} trappingagent. Maximum rates of HO{dot} production by thylakoids asindicated by the formation of methane sulfinic acid were observedunder aerobic conditions in the absence of added electron acceptors.They were higher than 2 µmol (mg Chl h)^–1. Saturationof HO{dot} production occurred at the low photon flux densityof 100 µmol m^–2 s^–1. Trapping of HO{dot} bydimethylsulfoxide suppressed, but did not eliminate light-dependentinactivation of PSI and II suggesting that HO{dot} formationcontributed to the photosensitivity of isolated thylakoids.DCMU inhibited HO{dot} formation. Importantly, methylviologendecreased HO{dot} formation in the absence, but stimulated itin the presence of Fe³⁺. In intact chloroplasts, HO{dot} formation became appreciableonly after KCN had been added to inhibit effective H₂O₂ scavengingby ascorbate peroxidase. It was stimulated by ferrisulfate,but not by ferricyanide which does not penetrate the chloroplastenvelope. Infiltrated spinach leaves behaved similar in principleto intact chloroplasts in regard to HO{dot} formation but HO{dot}production was very slow if detectable at all by the formationof methylsulfinic acid indicating effective radical detoxification. HO{dot} formation is interpreted to be the result of a Fenton-typereaction which produces HO{dot} in chloroplasts from H₂O₂ andreduced ferredoxin, when O₂ is electron acceptor in the Mehlerreaction and radical detoxification reactions are inhibited. (Received November 13, 1996; Accepted April 23, 1996)     

Further Characteristics of Salt-Dependent Bicarbonate Use by the Seagrass Zostera muelleri

Millhouse, J. and Strother, S. 1987. Further characteristicsof salt-dependent bicarbonate use by the seagrass Zostera muelleri.—J.exp. Bot. 38: 1055–1068. The contribution of HCO^–₃to photosynthetic O₂ evolutionin the seagrass Zostera muelleri Irmisch ex Aschers. increasedwith increasing salinity of the bathing seawater when the inorganiccarbon concentration was kept constant. K_1/2 (seawater salts)for HCO^–₃ -dependent photosynthesis was 66% of seawatersalinity. Both short- and long-term pretreatment at low salinitiesstimulated photosynthesis in full strength seawater. Twentyfour hours pre-incubation of seagrass plants in 3·0 molm^–3 NaHCO₃ resulted in increased photosynthesis at allsalinities, apparently due to stimulation of HCO^–₃ use(K_1/2 (seawater salts) = 26%). V_max (HCO^–₃) was not affectedby low salinity pretreatment. The kinetics of HCO^–₃ stimulationby the major seawater cations was investigated. Ca²⁺ was themost effective cation with the highest V_max (HCO^–₃) andwith K_1/2(Ca²⁺) = 14 mol m^–3. Mg²⁺ was also very effectiveat less than 50 mol m^–3 but higher concentrations wereinhibitory. This inhibition cannot be accounted for solely byprecipitation of MgCO₃. Na⁺ and K⁺ were both capable of stimulatingHCO^–₃ use. Stimulation was in two distinct parts. Up to500 mol m^–3, both citrate and chloride salts gave similarresults (K_1/2(Na⁺) 81 mol m^–3, V_max(HCO^–₃) 0·26µmol O₂ mg^–1 chl min^–1), but use of citratesalts above 500 mol m^–2 caused a second stimulation ofHCO^–₃ use (K_1/2(Na⁺) 830 mol m^–3, V_max(HCO^–₃)0·68 µmol O₂ mg^–1 chl min^–1). V_max(HCO^–₃)for the second-phase Na⁺ or K⁺ stimulation was of the same orderas for Ca²⁺-stimulated HCO^–₃ use. To further characterizesalt-dependent HCO^–₃ use, the sensitivity of photosynthesisto Tris and TES buffers was investigated. The effects of Trisappear to be due to the action of Tris⁺ causing stimulationof HCO^–₃ -dependent photosynthesis in the absence of salt,but inhibition of HCO^–₃ use in saline media. TES has noeffect on photosynthesis. External carbonic anhydrase, althoughimplicated in salt-dependent HCO^–₃ use in Z. muelleri,could not be detected in whole leaves. Key words: Zostera muelleri, HCO^–₃ use, salinity     

Role of carbonic anhydrase in photosynthesis in Chlorella derived from kinetic analysis of 14CO2 fixation1

Time courses of photosynthetic ¹⁴CO₂ fixation and its simulationare presented for Chlorella cells grown under low CO₂ concentration(low-CO₂ cells) and subsequently exposed to 0.2 mM NaH¹⁴CO₃or 130 ppm ¹⁴CO₂ in the presence or absence of carbonic anhydrase(CA) in the suspending medium. It was shown that Chlorella cells utilized only free CO₂ whenNaHCO₃ was given in the presence or absence of CA, or when CO₂was bubbled in the absence of CA. However, the present simulationindicated that both CO₃ and HCO₃^– were utilized when CO₂was given in the presence of CA. Based on these results, weconcluded that 1) Chlorella cells absorb only free CO₂ and 2)this gas is provided to algal cells in two ways, i.e., by directand indirect CO₂ supply. Usually, the dissolved CO₂ is directlyutilized by the algal cells (direct supply of CO₂). However,when the concentration of dissolved CO₂ is extremely low andwhen there is CA, CO₂ reconverted from HCO₃^– is also utilizedby Chlorella cells (indirect supply of CO₂). The utilizationof HCO₃^– indicated by the above simulation was explainedby the indirect supply of CO₂. We further assumed that the indirectsupply of CO₂ to ribulose 1,5-bisphosphate carboxylase occursmainly in the chloroplasts of low-CO₂ cells containing highCA. Thus, under low CO₂ concentrations, low-CO₂ cells can carryout more efficient CO₂ fixation than high-CO₂ cells, resultingin the lower apparent Km(CO₂). ³Department of Biology, Faculty of Science, Niigata University,Niigata, Japan. (Received April 2, 1980; )     

Interaction of Respiration, Ion Regulation, and Acid-Base Balance in the Everyday Life of Aquatic Crustaceans

Extracellular and intracellular acid-base balance is necessaryfor the maintenance of normal metabolic processes. The primarysource of acid is metabolically produced CO₂, and the CO₂/HCO₃^–system is the most significant buffer. The regulation of acid-basebalance is complex, involving the interaction between respiratorygas exchange and ion transport. In aquatic crustaceans respirationis governed by the need to extract oxygen from water, an O₂-poormedium; thus, acid-base balance is maintained primarily throughion transport mechanisms. These mechanisms include Na⁺/H⁺ andCl^–/HCO₃^– exchange processes that are sensitiveto the extracellular acid-base status of the animal. In marinecrabs, ion regulation and acid-base balance are accomplishedby the posterior gills, while in freshwater species all gillsand the antennal gland perform these functions. Intracellularacid-base balance appears to be maintained primarily by iontransport across the cell membrane. Hemolymph pH varies inverselywith acclimation temperature and salinity. In both cases Pco₂remains nearly constant, and the pH change is a result of changesin hemolymph HCO₃^– concentrations brought about by ionexchange mechanisms. Environmental hypercapnia or hyperoxiainduces a repiratory acidosis characterized by increased Pco₂,low pH, and elevated HCO₃^–; this is partially compensatedfor by ion exchange processes that bring about a further increasein hemolymph HCO₃^–. Exercise causes a mixed respiratoryand metabolic acidosis with compensation via H⁺ ion excretionand hyperventilation.