Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter

We have clonedand functionally characterized the human Na⁺-dependenthigh-affinity dicarboxylate transporter (hNaDC3) from placenta. ThehNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the clonedtransporter mediates the transport of succinate in the presence ofNa⁺ [concentration of substrate necessary for half-maximaltransport (K_t) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. TheNa⁺-to-succinate stoichiometry is 3:1 and concentration ofNa⁺ necessary for half-maximal transport(K^Na+_0.5) is 49 ± 1 mM as determined by uptake studies withradiolabeled succinate. When expressed in Xenopuslaevis oocytes, hNaDC3 induces Na⁺-dependent inwardcurrents in the presence of succinate and dimethylsuccinate. At amembrane potential of 50 mV,K^Suc_0.5 is 102 ± 20 µM andK^Na+_0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer andradiolabeled succinate uptake in hNaDC3-expressing oocytes indicate acharge-to-succinate ratio of 1:1 for the transport process, suggestinga Na⁺-to-succinate stoichiometry of 3:1. pH titration ofcitrate-induced currents shows that hNaDC3 accepts preferentially thedivalent anionic form of citrate as a substrate. Li⁺inhibits succinate-induced currents in the presence of Na⁺.Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The humanNaDC3 gene is located on chromosome20q12-13.1, as evidenced by fluorescent in situ hybridization. Thegene is >80 kbp long and consists of 13 exons and 12 introns.

     

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage

Ca²⁺-activatedCl currents (I_Cl,Ca) wereexamined using fluorescence confocal microscopy to monitorintracellular Ca²⁺ liberation evoked by flash photolysis ofcaged inositol 1,4,5-trisphosphate (InsP₃) involtage-clamped Xenopus oocytes. Currents at +40 mV exhibited asteep dependence on InsP₃ concentration([InsP₃]), whereas currents at140 mV exhibited a higher threshold and more graded relationshipwith [InsP₃]. Ca²⁺ levelsrequired to half-maximally activate I_Cl,Ca wereabout 50% larger at 140 mV than at +40 mV, and currents evokedby small Ca²⁺ elevations were reduced >25-fold. Thehalf-decay time of Ca²⁺ signals shortened at increasinglypositive potentials, whereas the decay of I_Cl,Calengthened. The steady-state current-voltage (I-V) relationshipfor I_Cl,Ca exhibited outward rectification withweak photolysis flashes but became more linear with stronger stimuli.Instantaneous I-V relationships were linear with both strongand weak stimuli. Current relaxations following voltage steps duringactivation of I_Cl,Ca decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at 160 mV. We conclude that InsP₃-mediated Ca²⁺liberation activates a single population of Clchannels, which exhibit voltage-dependent Ca²⁺ activationand voltage-independent instantaneous conductance.

     

Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells

Chloride release from nonpigmented ciliary epithelial (NPE)cells is a final step in forming aqueous humor, and adenosine stimulates Cl transport by these cells. Whole cell patchclamping of cultured human NPE cells indicated that theA₃-selective agonist1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl--D-ribofuranuronamide (IB-MECA) stimulated currents (I_IB-MECA) by~90% at +80 mV. Partial replacement of external Clwith aspartate reduced outward currents and shifted the reversal potential (V_rev) from 23 ± 2 mV to0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusionwith the Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acidinhibited the currents. Partial Cl replacement withaspartate and NO₃, and perfusion with NPPB, hadsimilar effects on the swelling-activated whole cell currents(I_Swell). Partial cyclamate substitution for external Cl inhibited inward and outward currents of bothI_IB-MECA and I_Swell. Bothsets of currents also showed outward rectification and inactivation atlarge depolarizing potentials. The results are consistent with theconcept that A₃-subtype adenosine agonists and swellingactivate a common population of Cl channels.

     

Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements

Members of the SLC20 family or type III Na⁺-coupled P_i cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P_i homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P_i cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na⁺-dependent ³²P_i uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li⁺ substituted for Na⁺. The dependence of the P_i-induced current on P_i concentration was Michaelian, and the dependence on Na⁺ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P_i affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual ²²Na-³²P_i uptake and suggested a 2:1 Na⁺:P_i stoichiometry. A correlation of ³²P_i uptake and net charge movement indicated one charge translocation per P_i. Changes in oocyte surface pH were consistent with transport of monovalent P_i. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na⁺:H₂PO₄^–:Na⁺. Significantly, in contrast to type II Na⁺-P_i cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na⁺-P_i cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor     

Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains

The solute carrier gene family SLC26consists of tissue-specific anion exchanger genes, three of themassociated with distinct human recessive disorders. By a genome-drivenapproach, several new SLC26 family members have been identified,including a kidney- and pancreas-specific gene, SLC26A6. We report thefunctional characterization of SLC26A6 and two new alternativelyspliced variants, named SLC26A6c and SLC26A6d. Immunofluorescencestudies on transiently transfected cells indicated membranelocalization and indicated that both NH₂- and COOH-terminaltails of the SLC26A6 variants are located intracellularly, suggesting atopology with an even number of transmembrane domains. Functionalexpression of the three proteins in Xenopus oocytesdemonstrated Cl and SO transportactivity. In addition, the transport of SO andCl was inhibited by DIDS and HCO. We demonstrated also that the COOH terminus of SLC26A6 binds to the firstand second PDZ domains of the Na⁺/H⁺ exchanger(NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor(NHERF) proteins in vitro. Truncation of the last three amino acids(TRL) of SLC26A6 abrogated the interaction but did not affect transportfunction. These results demonstrate that SLC26A6 and its two splicevariants can function as anion transporters linked to PDZ-interactionpathways. Our results support the general concept of microdomainorganization for ion transport and suggest a mechanism for cysticfibrosis transmembrane regulator (CFTR)-mediated SLC26A6 upregulationin pancreatic duct cells.

     

Activation of Ca2+-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells

The effects ofcyanide (CN) on whole cell current measured with the perforated-patchmethod were studied in adrenal medullary cells. Application of CNproduced initially inward and then outward currents at 52 mV ormore negative. As the membrane potential was hyperpolarized, amplitudeand latency of the outward current (I_o) by CNbecame small and long, respectively. A decrease in the externalNa⁺ concentration did not affectthe latency for CN-inducedI_o but enhancedthe amplitude markedly. The CNI_o reversedpolarity at 85 mV, close to the Nernst potential forK⁺, and was suppressed by theK⁺ channel blockers curare andapamin but not by glibenclamide, suggesting thatI_o is due to theactivation of Ca²⁺-dependentK⁺ channels. Consistent with thisnotion, the Ca²⁺-mobilizingagents, muscarine and caffeine, also producedI_o. Exposure toCN in a Ca²⁺-deficient medium for4 min abolished caffeine- or muscarine-induced I_o withoutdevelopment ofI_o, and additionof Ca²⁺ to the CN-containingsolution inducedI_o. We concludethat exposure to CN producesCa²⁺-dependentK⁺ currents in an externalCa²⁺-dependent manner, probablyvia facilitation of Ca²⁺ influx.

     

Cell cycle-related changes in transient K(+) current density in the GH3 pituitary cell line

Our aim was to determinewhether the expression of K⁺ currents is related to thecell cycle in the excitable GH3 pituitary cell line. K⁺currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cellcycle. We show that the peak density of the transient outward K⁺ current (I_to) was 33% lower incells in S phase (BrdU+) than in cells in other phases of the cellcycle (BrdU). The voltage-dependence of I_towas not modified. However, of the two kinetic components ofI_to inactivation, the characteristics of thefast component differed significantly between BrdU+ and BrdU cells.Recovery from inactivation of I_to showedbiexponential and monoexponential function in BrdU and BrdU+ cells,respectively. This suggests that the molecular basis of this currentvaries during the cell cycle. We further demonstrated that4-aminopyridine, which blocks I_to, inhibited GH3cell proliferation without altering the membrane potential. These datasuggest that I_to may play a role in GH3 cellproliferation processes.

     

Modulation of K+ currents in monocytes by VCAM-1 and E-selectin on activated human endothelium

Resting membrane potential (RMP) and whole cell currents wererecorded in human THP-1 monocytes adherent to polystyrene, unstimulated human umbilical vein endothelial cells (HUVECs),lipopolysaccharide (LPS)-treated HUVECs, immobilizedE-selectin, or vascular cell adhesion molecule 1 (VCAM-1)using the patch-clamp technique. RMP after 5 h on polystyrene was24.3 ± 1.7 mV (n = 42) with delayed rectifier K⁺(I_dr) andCl currents(I_Cl) presentin >75% of the cells. Inwardly rectifying K⁺ currents(I_ir) werepresent in only 14% of THP-1 cells. Adherence to unstimulated HUVECsor E-selectin for 5 h had no effect on I_ir orI_Cl but decreasedI_dr. Five hoursafter adherence to LPS-treated HUVECs, outward currents were unchanged,but I_ir waspresent in 81% of THP-1 cells. A twofold increase inI_ir and ahyperpolarization (41.3 ± 3.7 mV,n = 16) were abolished by pretreatmentof THP-1 cells with cycloheximide, a protein synthesis inhibitor, orherbimycin A, a tyrosine kinase inhibitor, or by pretreatment of theLPS-treated HUVECs with anti-VCAM-1. Only a brief (15-min) interactionbetween THP-1 cells and LPS-treated HUVECs was required toinduce I_ir expression 5 h later. THP-1 cells adherent to VCAM-1 exhibited similarconductances to cells adherent to LPS-treated HUVECs. Thus engagementof specific integrins results in selective modulation of differentK⁺ conductances.

     

Effect of Low Relative Humidity on {delta}13C Value in Two C3 Grasses and in Panicum milioides, a C3-C4 Intermediate Species

When grown under conditions of low relative humidity, the C₃–C₄intermediate Panicum milioides, as well as the C₃ grasses Triticumaestivum and Poa pratense, exhibited ¹³C values which were upto 2–7%o less negative than the ¹³C values of the correspondingplants grown at high relative humidity. At both humidity levels,there was no evidence of a substantial contribution of phosphoenolpyruvatecarboxylase to carbon gain in Panicum milioides     

Intracellular Cs+ activates the PKA pathway,revealing a fast,reversible, Ca2+-dependent inactivation of L-type Ca2+ current

Inactivation of the L-type Ca²⁺ current (I_CaL) was studied in isolated guinea pig ventricular myocytes with different ionic solutions. Under basal conditions, I_CaL of 82% of cells infused with Cs⁺-based intracellular solutions showed enhanced amplitude with multiphasic decay and diastolic depolarization-induced facilitation. The characteristics of I_CaL in this population of cells were not due to contamination by other currents or an artifact. These phenomena were reduced by ryanodine, caffeine, cyclopiazonic acid, the protein kinase A inhibitor H-89, and the cAMP-dependent protein kinase inhibitor. Forskolin and isoproterenol increased I_CaL by only 60% in these cells. Cells infused with either N-methyl-D-glucamine or K⁺-based intracellular solutions did not show multiphasic decay or facilitation under basal conditions. Isoproterenol increased I_CaL by 200% in these cells. In conclusion, we show that multiphasic inactivation of I_CaL is due to Ca²⁺-dependent inactivation that is reversible on a time scale of tens of milliseconds. Cs⁺ seems to activate the cAMP-dependent protein kinase pathway when used as a substitute for K⁺ in the pipette solution. L-type calcium current; calcium-dependent inactivation; facilitation; phosphorylation; cesium     

Characterization of G proteins involved in activation of nonselective cation channels and arachidonic acid release by norepinephrine/alpha1A-adrenergic receptors

We demonstrated recently that norepinephrine activates Ca²⁺-permeable nonselective cation channels (NSCCs) in Chinese hamster ovary cells stably expressing _1A-adrenergic receptors (CHO-_1A). Moreover, extracellular Ca²⁺ through NSCCs plays essential roles in norepinephrine-induced arachidonic acid release. The purpose of the present study was to identify the G proteins involved in the activation of NSCCs and arachidonic acid release by norepinephrine. For these purposes, we used U73122, an inhibitor of phospholipase C (PLC), and dominant negative mutants of G₁₂ and G₁₃ (G₁₂G228A and G₁₃G225A, respectively). U73122 failed to inhibit NSCCs activation by norepinephrine. The magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₃G225A were smaller than those in CHO-_1A. In contrast, the magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₂G228A were similar to those in CHO-_1A. In addition, neither a Rho-associated kinase (ROCK) inhibitor nor a phosphoinositide 3-kinase inhibitor affected norepinephrine-induced extracellular Ca²⁺ influx. G₁₃G225A, but not G₁₂G228A, also inhibited arachidonic acid release partially. These results demonstrate that 1) the G_q/PLC-pathway is not involved in NSCCs activation by norepinephrine, 2) G₁₃ couples with CHO-_1A and plays important roles for norepinephrine-induced NSCCs activation, 3) neither ROCK- nor PI3K-dependent cascade is involved in NSCCs activation, and 4) G₁₃ is involved in norepinephrine-induced arachidonic acid release in CHO-_1A. norepinephrine; _1A-adrenergic receptor; nonselective cation channel; G₁₃ protein; arachidonic acid release     

A comparison of the impacts of various nitrogen sources on acid-base balance in C3 Triticum aestivum L. and C4 Zea mays L. plants

This work aimed to study the impacts of acquisition and assimilationof various nitrogen sources, i.e. NO₃^–, NH₄⁺ or NH₄NO₃,in combination with gaseous NH₃ on plant growth and acid-basebalance in higher plants. Plants of C₃ Triticum aestivum L.and C₄ Zea mays L. grown with shoots in ambient air in hydroponicculture solutions with 2 mol m^–3 of nitrogen source asNO₃^–, NH₄⁺ or NH₄NO₃ for 21 d and 18 d, respectively,had their shoots exposed either to 320 µg m^–3 NH₃or to ambient air for 7 d. Variations in plant growth (leaves,stubble and roots), and OH^– and H⁺ extrusions as wellas the relative increases in nitrogen, carbon and carboxylatewere determined. These data were computed as H⁺/N, H⁺/C, (C-A)/N,and (C-A)/C to analyse influences of different nitrogen sourceson acid-base balance in C₃ Triticum aestivum and C₄ Zea maysplants. Root growth in dry weight gain was significantly reduced bytreatment with 320 µg m^–3 NH₃ in Triticum aestivumand Zea mays growing with different N-forms, whereas leaf growthwas not significantly affected by NH₃. In comparison with C₃Triticum aestivum, non-fumigated C₄ Zea mays had low ratiosof OH^–/N in NO₃^–3-grown plants and of H⁺/N in NH₄⁺- and NH ₄NO₃-grown plants. Utilization of NH₃ from the atmospherereduced both the OH^–N ratios in NO₃^– -grown plantsand the H⁺/N ratio in NH₄⁺ - and NH₄NO₃ -grown plants of bothspecies. Furthermore, Zea mays had higher ratios of (C-A)/Nin NH₄⁺ - and NH₄NO₃-grown plants than Triticum aestivum. Thismeans that C₄ Zea mays had synthesized more organic anion perunit increase in organic N than C₃ Triticum aestivum plants.Within both species, different nitrogen sources altered theratios of (C-A)/N in the order: NH₄NO₃>NH₄⁺>NO₃^–.Fumigation with NH₃ increased organic acid synthesis in NO₃^–- and NH₄⁺ - grown plants of Triticum aestivum, whereas it decreasedorganic acid synthesis in Zea mays plants under the same conditions.Furthermore, these differences in acid-base regulation betweenC₃ Triticum aestivum and C₄ Zea mays plants growing with differentnitrogen sources are discussed. Key words: Acid-base balance, ammonia, ammonium, nitrate, ammonium nitrate, C₃ Triticum aestivum L., C₄ Zea mays L.     

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers onfilters to analyze the apical membrane mechanisms that help mediate ionand fluid transport across the epithelium. RT-PCR showed the presenceof cystic fibrosis transmembrane conductance regulator (CFTR) andepithelial Na⁺ channel (ENaC) message, and immunomicroscopyshowed apical membrane staining for both proteins. CFTR was alsolocalized to the apical membrane of native human mammary ductepithelium. In control conditions, mean values of transepithelialpotential (apical-side negative) and resistance(R_T) are 5.9 mV and 829  · cm², respectively. The apical membranepotential (V_A) is 40.7 mV, and the mean ratioof apical to basolateral membrane resistance (R_A/R_B) is 2.8. Apicalamiloride hyperpolarized V_A by 19.7 mV andtripled R_A/R_B. AcAMP-elevating cocktail depolarized V_A by 17.6 mV, decreased R_A/R_B by60%, increased short-circuit current by 6 µA/cm²,decreased R_T by 155  · cm², and largely eliminated responses toamiloride. Whole cell patch-clamp measurements demonstratedamiloride-inhibited Na⁺ currents [linear current-voltage(I-V) relation] and forskolin-stimulated Clcurrents (linear I-V relation). A capacitance probe methodshowed that in the control state, MEC monolayers either absorbed orsecreted fluid (2-4µl · cm² · h¹). Fluidsecretion was stimulated either by activating CFTR (cAMP) or blockingENaC (amiloride). These data plus equivalent circuit analysis showedthat 1) fluid absorption across MEC is mediated byNa⁺ transport via apical membrane ENaC, and fluid secretionis mediated, in part, by Cl transport via apicalCFTR; 2) in both cases, appropriate counterions move throughtight junctions to maintain electroneutrality; and 3)interactions among CFTR, ENaC, and tight junctions allow MEC to eitherabsorb or secrete fluid and, in situ, may help control luminal[Na⁺] and [Cl].

     

Potassium Channels at Chara Plasmalemma

Exposure to high K⁺ medium transforms Chara plasmalemma into[K⁺]_osensitive state (K⁺ state). The current-voltage (I/V)characteristicsunder such conditions display a negative conductance region.This feature results from the complex time and voltage dependenceof K⁺ channel opening At potentials more negative than a thresholdp.d. the channels are closed and the I/V characteristics becomelinear with a low slope conductance of 0.8 S m² and only a weakdependence on [K⁺]_o. Such behaviour is usually associated witha non-specific leak current The threshold level for K⁺ channelclosing depends on [K⁺]_o. In 2.0 mol m^–3 and 5.0 mol m^–3K⁺ medium the membrane resting p.d. follows E_K, but hyperpolarizesgradually if the [K⁺]_o is lowered. The proton pump thus appearsto be non-operative, while the cell is in the K⁺ state, andrecovers slowly as the cell is returned to a low K⁺ medium.Excitation currents decline if the cells are kept in K⁺ statefor some hours. Key words: K⁺ channels, Chara corallina, Proton pump, Current/, oltage characteristics, Conductance     

Carbon Metabolism in Seagrasses: II. PATTERNS OF PHOTOS YNTHETIC CO2 INCORPORATION

Patterns of initial photosynthetic CO₂ incorporation were determinedfor some seagrasses and were related to activities of primarycarbon fixing enzymes, carbonic anhydrase activities, and ¹³Cvalues. According to the incorporation patterns, Cymodocea nodosa wasa C4 species while Thalassia hemprichli and Thalassodendronciliatum were C₃ plants. Halophila stipulacea showed an unusualincorporation pattern which could be viewed as intermediatebetween typical C₃ and C₄ pathways. The activity ratios of ribulose-l,5-bisphosphate carboxylase (RUBPcase) to phosphoenolpyruvatecarboxylase (PEPcase) were about 3 for Thalassodendron ciliatumand 1 for Cymodocea nodosa and Halophila stipulacea. The lattervalue, which is intermediate to ratios found in terrestrialC₃ and C₄ plants, may correlate with the incorporation patternsfound for Halophila stipulacea. Since the C₄ seagrass lackedthe Kranz anatomy, it may, in addition, point to a flexibleincorporation potential for these plants. The high ¹³C values found in these and other seagrasses didnot correlate with their photosynthetic pathways as in terrestrialplants. This discrepancy is probably due to a ‘closedsystem’ type of photosynthesis in which CO₂ is efficientlyutilized. The C₃ species which utilize CO₂ enzymatically must convertexogenous HCO-₃ to CO₂ internally. Even though carbonic anhydraseactivities were very low, conversion rates seemed to be sufficientfor high rates of photosynthesis. Since enzymatic fixation ratesapproached photosynthetic rates even at CO₂ saturation, thelimitation for these seagrasses to express their high photosyntheticpotential is most probably the HCO^–₃ uptake system.     

Respiratory Utilization of 13C-Labelled Photosynthate in Nodulated Root Systems of Soybean Plants

Kouchi, H., Akao, S. and Yoneyama, T. 1986. Respiratory utilizationof ¹³C-labelled photosynthate in nodulated root systems of soybeanplants.—J. exp. Bot. 37: 985–993. An improved method for the measurement of respiratory utilizationof current photosynthate in the nodulated root system of water-culturedsoybean (Glycine max L.) plants was developed using a steady-state¹³CO₂ labelling technique. Well-nodulated plants at the latevegetative stage were allowed to assimilate ¹³CO₂ for 10 h incontinuous light at a constant CO₂ concentration with a constant¹³C abundance. The respiratory evolution of ¹³CO₂ from rootsand nodules was measured continuously throughout the periodof ¹³CO₂ assimilation and during a subsequent 36 h chase periodby using a differential infrared ¹³CO₂ analyser. The plantswere grown with nitrogen-free or (15 mmol dm^–3)-containing culture solution for 3 d before¹³CO₂ assimilation. In plants grown without , nodule respiration averaged 69% of the total respiration of the undergroundparts over the full experimental period and the CO₂ respiredreached an apparent isotopic equilibrium at 80–85% labellingafter initiating ¹³CO₂ assimilation. By contrast, the CO₂ respiredfrom the roots did not reach an isotopic equilibrium and labellingwas only 56% at the end of exposure to ¹³CO₂ These findingsdemonstrated that nodule respiration is strongly dependent onrecently assimilated carbon compared with root respiration. Plants supplied with in the culture solution showed a decreased rate of nodule respirationand a slightly increased rate of root respiration. The extentsand time courses of labelling of respired CO₂ from both theroots and nodules were similar in the presence and absence of except that the maximum level of labelling of CO₂ derived from nodule respiration in plantswith was significantly higher (about 91%) than for plants growing without . Key words: Soybean (Glycine max L.), nodule respiration, ¹³CO₂, assimilation, carbon partitioning     

Oxidative phosphorylation and the electron transport system of castor bean mitochondria

The difference spectrum (reduced minus oxidized) of castor bean(Ricinus communis L.) mitochondria showed the presence of cytochromeoxidase (cytochromes a+a₃), b-type cytochromes and cytochromec. The mitochondria actively oxidized succinate, -ketoglutarate,pyruvate and exogenous NADH, and oxidations of these substrateswere stimulated by added ADP, as in mammalian mitochondria.Values for the P/O ratio obtained for succinate, pyruvate and-ketoglutarate were the same as those reported for mammalianmitochondria, indicating that theoretical values are 2, 3 and4, respectively. The theoretical P/O ratio for exogenous NADHseemed to be 2. Oxidations of succinate and exogenous NADH instate 3 were almost completely inhibited by 0.3 mM cyanide and10 µM its antimycin A, while those of NAD⁺-linked substratesin state 3 were not completely suppressed even by excess concentrationsof these inhibitors. There seem to be two types of pathway forelectron transfer in the oxidation of NAD⁺-linked substratesin castor bean mitochondria, i.e. pathways which are sensitiveand insensitive to these inhibitors. Oxidation of exogenousNADH in state 3 was not inhibited by rotenone. Transitions of redox levels of the respiratory components fromstate 4 to state 3 on addition of ADP and from state 3 to state4 on exhaustion of added ADP were observed with a dual-wavelengthspectrophotometer. Effects of inhibitors on redox levels ofthe respiratory components in state 3 were investigated. Cytochromesof b-type and cytochrome c were fully reduced on addition ofcyanide. Cytochromes of b-type were also fully reduced on additionof antimycin A, but cytochrome oxidase (cytochromes a + a₃)and cytochrome c changed to the oxidized forms. The redox levelof the component(s) with an absorption maximum at 465 mµshifted further, but not completely, to the reduced side onaddition of antimycin A. However, this component(s) was oxidizedon addition of cyanide. Cyanide-, or antimycin A-resistant oxidationof NAD⁺-linked substrates seems to occur via an alternate electrontransfer pathway branching from NAD⁺-linked flavoprotein(s)in the mitochondria, not via the normal pathway through thecytochromes-cytochrome oxidase system. (Received June 8, 1970; )     

Hypothesis: do voltage-gated H(+) channels in alveolar epithelial cells contribute to CO(2) elimination by the lung?

Although alveolar epithelial cells were the firstmammalian cells in which voltage-gatedH⁺ currents were recorded, nospecific function has yet been proposed. Here we consider whetherH⁺ channels contribute to one ofthe main functions of the lung: CO₂ elimination. This idea buildson several observations: 1) somecell membranes have low CO₂permeability, 2) carbonic anhydrase is present in alveolar epithelium and contributes toCO₂ extrusion by facilitatingdiffusion, 3) the transepithelialpotential difference favors selective activation ofH⁺ channels in apical membranes,and 4) the properties ofH⁺ channels are ideally suited tothe proposed role. H⁺channels open only when the electrochemical gradient forH⁺ is outward, impartingdirectionality to the diffusion process. Unlike previous facilitateddiffusion models, HCO₃ andH⁺ recombine to formCO₂ in the alveolar subphase.Rough quantitative considerations indicate that the proposed mechanismis plausible and indicate a significant capacity forCO₂ elimination by the lung bythis route. Fully activated alveolarH⁺ channels extrude acidequivalents at three times the resting rate ofCO₂ production.

     

Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra

Freshly dispersedinterstitial cells from the rabbit urethra were studied by using theperforated-patch technique. When cells were voltage clamped at 60 mVand exposed to 10 µM norepinephrine (NE) at 80-s intervals, eitherlarge single inward currents or a series of oscillatory inward currentsof diminishing amplitude were evoked. These currents were blocked byeither phentolamine (1 µM) or prazosin (1 µM), suggesting that theeffects of NE were mediated via ₁-adrenoceptors.NE-evoked currents were depressed by the blockers ofCa²⁺-activated Cl currents, niflumic acid (10 µM), and 9-anthracenecarboxylic acid (9-AC, 1 mM). The reversalpotential of the above currents changed in a predictable manner whenthe Cl equilibrium potential was altered, againsuggesting that they were due to activation of a Clconductance. NE-evoked currents were decreased by 10 µM cyclopiazonic acid, suggesting that they were dependent on store-releasedCa²⁺. Inhibition of NE-evoked currents by the phospholipaseC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate(100 µM) suggested that NE releases Ca²⁺ via an inositol1,4,5-trisphosphate (IP₃)-dependent mechanism. Theseresults support the idea that stimulation of₁-adrenoceptors releases Ca²⁺ from anIP₃-sensitive store, which in turn activatesCa²⁺-activated Cl current in freshlydispersed interstitial cells of the rabbit urethra. This elevates slowwave frequency in these cells and may underlie the mechanismresponsible for increased urethral tone during nerve stimulation.

     

Temperature dependence of cloned mammalian and salmonid cardiac Na(+)/Ca(2+) exchanger isoforms

The cardiacNa⁺/Ca²⁺ exchanger (NCX), an importantregulator of cytosolic Ca²⁺ concentration in contractionand relaxation, has been shown in trout heart sarcolemmal vesicles tohave high activity at 7°C relative to its mammalian isoform. Thisunique property is likely due to differences in protein structure. Inthis study, outward NCX currents (I_NCX) of thewild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressedin oocytes were measured to explore the potential contributions ofregulatory vs. transport mechanisms to this observation. cRNA wastranscribed in vitro from both wild-type cDNA and was injected intoXenopus oocytes. I_NCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30°C using the giant excised patch technique. TheI_NCX for both isoforms exhibitedNa⁺-dependent inactivation and Ca²⁺-dependentpositive regulation. The I_NCX of NCX1.1exhibited typical mammalian temperature sensitivities withQ₁₀ values of 2.4 and 2.6 for peak and steady-statecurrents, respectively. However, the I_NCX ofNCX-TR1.0 was relatively temperature insensitive with Q₁₀values of 1.2 and 1.1 for peak and steady-state currents, respectively.I_NCX current decay was fit with a singleexponential, and the resultant rate constant of inactivation () wasdetermined as a function of temperature. As expected,  decreasedmonotonically with temperature for both isoforms. Although  wassignificantly greater in NCX1.1 compared with NCX-TR1.0 at alltemperatures, the effect of temperature on  was not differentbetween the two isoforms. These data suggest that thedisparities in I_NCX temperature dependencebetween these two exchanger isoforms are unlikely due to differences intheir inactivation kinetics. In addition, similar differences intemperature dependence were observed in both isoforms after-chymotrypsin treatment that renders the exchanger in a deregulatedstate. These data suggest that the differences in I_NCX temperature dependence between the twoisoforms are not due to potential disparities in either theI_NCX regulatory mechanisms or structuraldifferences in the cytoplasmic loop but are likely predicated ondifferences within the transmembrane segments.