Cellular Mechanisms of Organic Anion Transport in Crustacean Renal Tissue

Decapod Crustacea appear to utilize two overall strategies forexcretion of anionic wasteproducts and xenobiotics. One strategyutilizes a potent renal secretory system; the other utilizesboth modest renal secretion and strong gut secretion (possiblyaided by midgut gland conjugation). In crabs, the level of participationof the renal system in overall excretion is primarily determinedbythe location of an anion pump and a carrier protein. The bladder of Cancer borealis, which strongly secretes themodel organic anion, p-aminohippuric acid (PAH), has a serosalorganic anion pump and a luminal facilitated carrier. The bladderof Cancer trroratus, which reabsorbs PAH from bladder urine,has a luminal organic anion pump and a serosal facilitated carrier.     

Uptake of Radioactivity by Aquatic Plants and Location in the Cells I. THE EFFECT OF pH ON THE STRONTIUM-90 AND YTTRIUM-90 UPTAKE BY THE GREEN ALGA ULVA LACTUCA AND THE EFFECT OF STABLE YTTRIUM ON YTTRIUM-90 UPTAKE

The uptake of strontium-90 and yttrium-90 by Ulva lactuca fromsea water was measured over a period of 179 days. In sea waterat normal pH (8.0) and without added carrier Ulva reached astrontium-90 equilibrium at 0.32 times the concentration inthe water within 3 hrs, but rapidly depleted the water of yttrium-90.Addition of 0.15 ppm of non-radioactive (stable) yttrium ledto the establishment of an yttrium-90 equilibrium within 3 hrs,with the Ulva having a concentration factor of 550. It was foundthat without added carrier the yttrium-90 remained on the outersurface of the plants, but when carrier was added the yttrium-90taken up mainly entered the cells and was laid down in the protoplasmicinclusions. The strontium-90 was located only in the protoplasmicinclusion. Uptake was investigated in a pH range 5.1 to 8.0.Progressive reductions of pH from 8.0 to 6.5 led to a smallincrease in the amount of strontium-90 uptake but to large increasein the yttrium-90 uptake. However, in the pH range 5.8 to 5.1the yttrium-90 uptake was greatly reduced. External pH below6.0 caused death of the alga within 5 days. The effect of pHon strontium uptake is thought to be due to changes in equilibriumin the carbonate-bicarbonate system. The effects of pH and stableyttrium concentration on yttrium-90 uptake are discussed interms of changes in the state of yttrium in the sea water, resultingin increased availability to the plant, and of changes in thenature of the plant surface resulting from its physiologicalresponse to lowered pH.     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms     

The Specificity of the Carrier-Mediated Uptake of ABA by Root Segments of Phaseolus coccineus L.

Earlier work has established that the saturable component ofuptake of RS-[2¹⁴C]ABA by bean (Phaseolus coccineus L. cv. Prizewinner)root segments can be attributed to the action of a carrier.We now show that the carrier-mediated uptake is unaffected byRS-2-trans-ABA and lunularic acid and the unnatural R-ABA alsoappears to be ineffective. The specificity for S-ABA requiresthe halving of the K_m value for ABA determined previously (2.6mmol m_-3 for RS-; 1.3 mmol m_-3 for S-ABA). The RS-1', 4'-cis-dioland RS-1'-deoxy ABA reduce the uptake of RS-[2-₁₄C]ABA aboutas strongly as does unlabelled ABA, the K¹ for 1'-deoxy ABAwas similar to the K_m for ABA. The K₁ for RS-1', 4'-trans-diolwas 15.7 mmol m_-3. Consideration of the stereochemistry of thesecompounds suggests that the face of the ring of ABA away fromthe 1'-hydroxyl group interacts with the carrier site. Labelled material diffused out of undamaged root surfaces whichhad absorbed RS-[³H]ABA through an apical cut, suggesting thatABA is present in the apoplast. A simplified hypothesis is presented that can account for polartransport of ABA based on a gradient of a carrier in a tissuebut where the carrier is distributed uniformly on the apicaland basal ends of each cell. Key words: Uptake carrier, Abscisic acid, 1', 4'-Diol, Lunularic acid, Phaseolus coccineus, Polar-transport, Deoxyabscisic acid     

A Comparative Study of the Biological Activities of Two Molecular Species of Chloroplast-type Ferredoxins

Pairs of two molecular species of soluble chloroplast-type ferredoxins(Fd I and Fd II) from Nostoc muscorum and Aphanothece sacrumwere used to examine and compare the abilities of ferredoxinto substitute for spinach ferredoxin in the photoreduction ofNADP⁺ by spinach chloroplasts or N. muscorum membrane fragmentsand to link the reducing power of illuminated spinach chloroplaststo the Bacillus polymyxa nitrogenase system. Ferredoxins II of Nostoc and Aphanothece showed rather low activitiesin NADP⁺ photoreduction and nitrogenase system with spinachchloroplasts as the photosensitizer, compared to other ferredoxins.However, there was no difference between two ferredoxins (FdI and Fd II) from Nostoc in NADP⁺ photoreduction by photosyntheticmembrane fragments prepared from the same organism, N. muscorum. The biological significance of two molecular species of ferredoxinsin one organism could be ascribed to the different contributionof each ferredoxin to certain biological reactions in whichferredoxin functioned as an electron carrier. (Received November 4, 1980; Accepted January 9, 1981)     

The Effects of Oil-based Fungicides on Water Loss of Coleus blumei Leaves and Pelargonium Hybrid Petals

Effects of oil-based fungicides on plant surfaces were studiedby assessing water loss from excised sprayed leaves or petalsand by microscopy. Two fungicides (Turbair Zineb Fungicide andTurbair Copper Fungicide) were sprayed, using a Potter tower,onto the adaxial surfaces of excised leaves or petals, and weightloss was determined as a measure of water loss. Doses of approximately4–9 g m^–2 significantly increased water loss fromPelargonium hybrid petals and leaves of Coleus blumei Benth.The fungicides, their supernatants and the carrier oil increasedwater loss from the petals but effects of the supernatants andthe carrier oil on C. blumei leaves were less conclusive. LowTemperature Scanning Electron Microscopy (LTSEM) did not revealany scoring or marked erosion of the adaxial surface of leavesor petals 24 h after application of the fungicides. Coleus blumei Benth., Pelargonium hybrid, cuticle, fungicides, oils, LTSEM, water loss     

Nature of light-induced absorbance changes at 682 m{micro} and 702 m{micro} in photosynthesis of Anacystis nidulans

Light-induced absorbance changes in the region around the redabsorption band of chlorophyll a were measured in cells andlamella fragments of Anacystis nidulans. In both materials,absorbance decreases were observed at 702 mµ and 682 mµ.(The pigments are designated as P₇₀₀ and P₆₈₀.) The nature ofP₆₈₀ was investigated with special reference to its relationshipto P₇₀₀. In the cells, light absorbed by chlorophyll a causedan absorbance decrease at 682 mµ; Simultaneous illuminationwith light absorbed by phycocyanin caused a partial recoveryof the absorbance decrease. Similar results were observed withthe light-induced absorbance change at 702 mµ. This indicatesthat P₆₈₀ is also an electron carrier in the electron transportchain and occupies a place between the two photoreactions. Inlamella fragments, both the light-induced reversible absorbancechanges of P₆₈₀ and P₇₀₀ appeared in the presence of an electrondonor system; i.e., ascorbate and 2,6-dichlorophenolindophenolor N,N,N',N'-tetramethyl-l,4-phenylenediamine. The experimentsin which the oxidation-reduction potential of the reaction mediumwas changed showed that both P₆₈₀ and P₇₀₀ are one-electroncarriers, having a normal oxidation-reduction potential of 0.44v (assuming that the normal oxidation-reduction potential ofthe ferricyanide-ferrocyanide system is 0.409 v). A possibilitywas suggested that the absorbance change observed at 682 mµis another expression of the oxidation-reduction reaction ofP₇₀₀). (Received October 30, 1968; )     

Glutamine transport by the blood-brain barrier: a possible mechanism for nitrogen removal

Glutamine and glutamate transport activities were measuredin isolated luminal and abluminal plasma membrane vesiclesderived from bovine brain endothelial cells. Facilitativesystems for glutamine and glutamate were almost exclusivelylocated in luminal-enriched membranes. The facilitativeglutamine carrier was neither sensitive to2-aminobicyclo(2,2,1)heptane-2-carboxylic acid inhibition nor did itparticipate in accelerated amino acid exchange; it therefore appearedto be distinct from the neutral amino acid transport system L1. TwoNa-dependent glutamine transporters were found in abluminal-enrichedmembranes: systems A and N. System N accounted for ~80% ofNa-dependent glutamine transport at 100 µM. Abluminal-enriched membranes showed Na-dependent glutamate transport activity. The presence of 1) Na-dependent carrierscapable of pumping glutamine and glutamate from brain into endothelialcells, 2) glutaminase withinendothelial cells to hydrolyze glutamine to glutamate and ammonia, and3) facilitative carriers forglutamine and glutamate at the luminal membrane may provide a mechanismfor removing nitrogen and nitrogen-rich amino acids from brain.

     

Carrier-Mediated ABA Uptake by Suspension-Cultured Phaseolus coccineus L. Cells: Stereospecificity and Inhibition by Ionones and ABA Esters

Astle, M. and Rubery, P. 1987. Carrier-mediated ABA uptake bysuspension-cultured Phaseolus coccineus L. cells: Stereospecificityand inhibition by ionones and ABA esters.—J. exp. Bot.38: 150–163. The substrate for the abscisic acid (ABA) carrier in Phaseoluscoccineus L. suspension-cultured cells is shown to be the (S)ABAenantiomer, K_m = 1?0 mmol m^–3. The methyl (MeABA) andphenyl (PheABA) esters of ABA inhibit carrier-mediated uptakeof ABA with half-maximal inhibition achieved at about 7?0 mmolm^–3 and 10 mmol m^–3 respectively: with (S)MeABAthis value is decreased to about 2?0 mmol m^–3. There isno demethylation of radioactive MeABA by the cells during 5min incubations. Although MeABA reversibly inhibits the ABAcarrier, it is not a transport substrate: association of radioactiveMeABA with living cells is unaffected by non-radioactive MeABAor ABA and, by comparison with frozen-and-thawed cells, it isshown that the radioactivity remains extracellular. It is proposedthat MeABA binds to the carrier to form an abortive complexthat is not translocated. The terpenoid ABA analogue LAB 144143also inhibits carrier-mediated ABA uptake. At concentrationsup to about 20 mmol m^–3 - and ß-ionone specificallyinhibit the ABA carrier with the half-maximal effect at about0?6 mmol m^–3 ß-ionone. However, at higher iononeconcentrations, the uptake of ABA, indol-3-yl acetic acid andof 5,5-dimethyloxazolidine-2,4-dione (DMO) are all stimulated:this may reflect general permeabilization of the membrane toweak acids by ionone. Key words: Uptake carrier, abscisic acid, methyl and phenyl esters of ABA, ionone, Phaseolus coccineus L. suspension culture     

Mechanism and regulation of folate uptake by human pancreatic epithelial MIA PaCa-2 cells

After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1) temperature and energy dependent; 2) pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3) Na⁺ independent; 4) saturable as a function of substrate concentration (apparent K_m = 0.762 ± 0.10 µM); 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake. human reduced folate carrier; small interfering RNA; transport regulation     

The Phloem Translocation of Enantiomeric Pairs of Weak Acids in Ricinus communis

A possible role for specific carrier mechanisms in the phloemtranslocation of xenobiotics in Ricinus communis L. var. Gibsonii(Nichols) has been investigated by comparing the phloem transportof enantiomers of three acidic compounds. No differences intranslocation were found between the R– and S-enantiomersof phenylalanine, 2-methoxy-2-phenylpropanoic acid or 5-methyl-5-phenyloxazolidine-2,4-dione.These similarities between transport of enantiomers indicatethat stereospecific carriers do not occur, even for the endogenousamino acid phenylalanine of which only one enantiomer occursnaturally in phloem sap. Whilst transport of the enantiomerswas similar, there were differences between the mobility ofthe three compounds, and these could largely be explained interms of their physicochemical properties, without the needto invoke the existence of specific carrier mechanisms. Key words: Phloem transport, enantiomers, Ricinus communis, weak acids     

Uptake of Lysine by Wild-Type and S-(2-Aminoethyl)-L-cysteine-Resistant Suspension-Cultured Cells of Triticum aestivum

The kinetics of uptake of L-lysine in wheat (Triticum aestivumcv. Chinese Spring) were analyzed in wild-type cells and inAEC-1 variant cells that are resistant to S-(2-aminoethyl)-L-cysteine(AEC). Uptake of lysine by AEC-1 cells was considerably slowerthan that by the wild-type cells. In the presence of carbonylcyanidem-chlorophenylhydrazone, the rates of uptake by both types ofcell were reduced to a similar linear component. Fitting theuptake data to one linear (diffusional) component and one Michaelis-Menten(active) system showed that, as compared to wild-type cells,AEC-1 cells have a reduced V_max and an increased K_m with respectto the active component, byt they have a similar diffusionalcomponent. Inhibition experiments with various amino acids indicatedthat the active component represents a carrier specific forbasic amino acids, which was competitively inhibited by AEC.The AEC-1 cells also showed reduced uptake of several neutraland acidic amino acids, but the rate of uptake of 3-O-methylglucosewas somewhat higher than that by wild-type cells. (Received May 16, 1989; Accepted September 4, 1989)     

Supplementation of the protist Chilomonas paramecium with a highly unsaturated fatty acid enhances its nutritional quality for the rotifer Keratella quadrata

We tested whether a fatty acid supplementation technique usingbovine serum albumin (BSA) as a carrier, previously developedfor autotrophic protists, is also appropriate for supplementationof Chilomonas paramecium—a flagellated heterotrophic protist.Chilomonas paramecium was successfully enriched with eicosapentaenoicacid (EPA) and docosahexaenoic acid (DHA), both known to beessential for crustacean zooplankton. Preparing C. parameciumenriched with EPA and DHA in concentrations similar to thosefound in Cryptomonas phaseolus, an alga known to support highgrowth and reproduction of Keratella, allowed us to test thedirect nutritional effects of EPA and DHA on population growthand reproduction of the rotifer Keratella quadrata. Growth ratesand egg production were highest when K. quadrata was fed withC. phaseolus. Compared to non-supplemented C. paramecium, eggproduction of K. quadrata was significantly enhanced on a dietof C. paramecium enriched with DHA, whereas no significant effectscould be attributed to EPA enrichment. We conclude that DHAis important for reproduction of K. quadrata but cannot explainthe majority of the difference in food quality between C. parameciumand C. phaseolus.     

Guanine nucleotide transport by atractyloside-sensitive and -insensitive carriers in isolated heart mitochondria

Inprevious work (McKee EE, Bentley AT, Smith RM Jr, and Ciaccio CE,Biochem Biophys Res Commun 257: 466-472, 1999), the transport of guanine nucleotides into the matrix of intact isolated heart mitochondria was demonstrated. In this study, the time course andmechanisms of guanine nucleotide transport are characterized. Twodistinct mechanisms of transport were found to be capable of movingguanine nucleotides across the inner membrane. The first carrier wassaturable, displayed temperature dependence, preferred GDP to GTP, anddid not transport GMP or IMP. When incubated in the absence ofexogenous ATP, this carrier had a V_max of946 ± 53 pmol · mg¹ · min¹ with aK_m of 2.9 ± 0.3 mM for GDP. However,transport of GTP and GDP on this carrier was completely inhibited byphysiological concentrations of ATP, suggesting that this carrier wasnot involved with guanine nucleotide transport in vivo. Becausetransport on this carrier was also inhibited by atractyloside, thiscarrier was consistent with the well-characterized ATP/ADP translocase. The second mechanism of guanine nucleotide uptake was insensitive toatractyloside, displayed temperature dependence, and was capable oftransporting GMP, GDP, and GTP at approximately equal rates but did nottransport IMP, guanine, or guanosine. GTP transport via this mechanismwas slow, with a V_max of 48.7 ± 1.4 pmol · mg¹ · min¹ and aK_m = 4.4 ± 0.4 mM. However, becausethe requirement for guanine nucleotide transport is low in nondividingtissues such as the heart, this transport process is neverthelesssufficient to account for the matrix uptake of guanine nucleotides andmay represent the physiological mechanism of transport.

     

Metabolic role of flavoprotein in terminal oxidation by cytochrome o from Vitreoscilla

NADH-cytochrome o reductase is associated with purified preparationsof cytochrome o, and these preparations can be separated into"reductase-enriched" and "reductase-poor" fractions by columnchromatography. Direct evidence for the presence of flavin inthese preparations was obtained from fluorescence spectra, andthe intensity of the fluorescence maxima was greater in reductase-enrichedpreparations of cytochrome o than in reductase-poor ones. Exogenouslyadded flavin stimulated the rate of NADH oxidation by molecularoxygen that is catalyzed by preparations of cytochrome o, morestimulation being observed with "reductase-poor" than with "reductase-enriched"preparations. Since reduction of cytochrome o in an aerobicsolution was also stimulated by added flavin, the primary effectof the latter is on the NADH-cytochrome o reductase side ofthe cytochrome. Possible explanations for the observed stimulationof the reduction of cytochrome o in aerobic solutions in thepresence of exogenous flavin are 1) reconstitution of flavin-deficientreductase, 2) flavin acting as a mobile electron carrier betweenthe reductase and the cytochrome, 3) cytochrome o being reducedby superoxide anion generated as an intermediate in the reactionof reduced flavin with oxygen. More direct evidence for theparticipation of a flavor-protein in the reduction of cytochromeo was the observed photochemical reduction of cytochrome o inan anaerobic CO atmosphere without added flavin using EDTA asan electron donor. (Received July 18, 1977; )     

Characterization of an abscisic acid carrier in suspension-cultured barley cells

The uptake of [³H]-abscisic acid in barley (Hordeum vulgareL. cv. Heartland) cell cultures was found to be mediated throughboth non-saturable and saturable components. The kinetic parametersof the saturable component, determined at pH 4.5 and 21 °C,showed a K_m for natural or (+ )-ABA of 1.3±0.7µMand an apparent V_mex of 7.0 ± 2.8 nmol g^–1 cellsh^–1. The carrier showed a strong preference for the naturalenantiomer of ABA as compared to the unnatural one. Other substancestested, e.g. amino acids, organic acids, and other growth regulators,did not appear to interfere with the carrier-mediated uptakeof ABA. At low external concentrations of ABA (below 2.0 µM),the saturable component was greater than the diffusion component.Similarly, between pH 4.0 and 6.0, the saturable uptake wasresponsible for more than 50% of the total uptake. The carriermay be important in vivo for mediating uptake when endogenouslevels of ABA are low (c. 1 µM). The carrier specificity was evident in inhibition experimentsdone with ABA analogues. Our data showed that the carrier couldaccommodate small modifications in the ABA structure. Four analogueswere able to compete efficiently with ( + )-ABA for the bindingsite of the carrier. Three of these competitors were of the(+)-series. Only one ( –)-analogue, (–)-ABA, wasable to inhibit markedly the saturable uptake of ( + )-ABA.The induction of the ABA-respons-ive gene WCS120 (Houde et al.,1992) presented stricter requirements for the ABA molecule thanthe carrier, although with a similar preference for the ( +)-analogues. Besides ( + )-ABA itself, only two of the analoguestested, both ( + )-series, were able to induce the WCS120 geneafter a 24 h incubation period. The absence of correlation betweenthe activity of the analogues as ABA inhibitors in the carriersystem, and their capacity to induce the WCS120 gene tend tosuggest that the carrier is not directly involved in the signaltransduction pathway leading to the induction of this specificgene. Key words: Abscisic acid, barley, gene induction, Hordeum vulgare, uptake carrier     

Studies on the Hill reaction of membrane fragments of blue-green algae II. The reaction steps inactivated at high water concentration

DPIP-photoreduction by membrane fragments of Anabaena cylindricaand A. variabilis was studied to determine which step(s) ofthe Hill reaction system is inactivated on incubation of themembrane fragments in a medium with a high water concentration(cf. 1). Supplementary experiments were done with Anacystisnidulans and Plectonema boryanum. After inactivation of the Hill system at a high water concentration,DPIP-photo-reducing activity was strongly enhanced in the A.variabilis system but less so in the A. cylindrica system byadding DPC, NH₂OH, Mn⁺⁺ or H₂0₂. The activity supported by theadded electron donor was inhibited by DCMU. The steady statelevel of chlorophyll fluorescence was lowered by the inactivationtreatment. In the A. variabilis system, the fluorescence yieldincreased to the original level on the addition of an electrondonor. In the A. cylindrica system, the yield was not so stronglyenhanced as in the A. variabilis system. We inferred that, in A. variabilis, inactivation occurs in thereaction system before the site which receives electrons fromartificial donors, probably including the water oxidation system.In A. cylindrica, besides this site, a site at or near the photochemicalsystem is also blocked. Similar types of inactivation were observed in DPIP-Hill reactionsusing Anacystis nidulans and Plectonema boryanum preparations.The characteristic stability of the Hill reaction system observedin two Anabaena preparations is probably common to the blue-greenalgae. (Received December 10, 1971; )     

Studies of sulfate utilization by algae 17. Reactions of the adenosine 5'-phosphosulfate (APS) sulfotransferase from Chlorella and studies of model reactions which explain the diversity of side products with thiols

Adenosine 5'-phosphosulfate sulfotransferase has been partiallypurified from Chlorella and is shown to catalyze the transferof the sulfate group of adenosine 5'-phosphosulfate to a varietyof thiol acceptors to form the corresponding organic thiosulfate.While the normal acceptor in the sulfate reducing pathway isthought to be a peptide carrier containing a thiol group theenzyme is very non-specific with respect to the thiols to whichit will transfer leading to a large number of side reactionswhich are possible when thiols are added to the system. Usingadenosine 5'-phosphosulfate and the enzyme, monothiols formsulfite and the organic thiosulfate of the thiol, with dithiolswhich readily form intramolecular disulfides, sulfite is theonly product, while with vicinal dithiols, sulfite and finallythiosulfate is formed. The -SO₃^– sulfur of the thiosulfateoriginates from adenosine 5'-phosphosulfate while the -S^–-sulfur is supplied by the vicinal dithiol. The same productscan be obtained using glutathione-S-sulfonate in place of adenosine5'-phosphosulfate and the enzyme, in a non-enzymatic reactionwith the same thiols. Thus it appears that the enzymatic reactioncatalyzes the transfer of the sulfate group of adenosine-5'-phosphosulfateto a thiol carrier or to any other thiol. When these other thiolsare present, however, sulfite, thiosulfate or organic thiosulfatesof the thiols are formed in non-enzymatic side reactions. Thetransferase from Chlorella is specific for adenosine 5'-phosphosulfateand will not catalyze the reaction with adenosine-3'-phosphate-5'-phosphosulfate. ¹Supported by Grants GB 4321, GB 40856X and BMS 73 00987 AO1from the National Science Foundation. ²Supported by a Gillette Graduate Fellowship. Portions of thispaper formed part of a dissertation presented to the graduatefaculty of Brandeis University in partial fulfillment of thePh.D. Degree. (Received June 30, 1976; )     

A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway

Vitamin B₆ is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B₆ and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B₆ homeostasis. Due to the water-soluble nature of vitamin B₆ and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B₆ in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na⁺ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (K_m) of 11.99 ± 1.41 µM and a maximal velocity (V_max) of 67.63 ± 3.87 pmol · mg protein^-1 · 3 min^-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (K_i) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca⁺²/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the V_max, but not the apparent K_m, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH dependent and amiloride sensitive and appears to be under the regulation of an intracellular PKA-mediated pathway. vitamin B₆; intestinal transport; transport regulation; Caco-2 cell     

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes

We recently identified a cDNA clone frommouse small intestine, which appears to be involved in folate transportwhen expressed in Xenopus oocytes. Theopen reading frame of this clone is identical to that of the reducedfolate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley,and K. H. Cowan. J. Biol. Chem. 269: 17-20,1994). The characteristics of this cDNA clone [previously referred toas intestinal folate carrier 1 (IFC-1)] expressed inXenopus oocytes, however, were foundto be different from the characteristics of folate transport in nativesmall intestinal epithelial cells. To further study these differences,we determined the characteristics of RFC when expressed in anintestinal epithelial cell line, IEC-6, and compared the findings toits characteristics when expressed inXenopus oocytes. RFC was stablytransfected into IEC-6 cells by electroporation; its cRNA wasmicroinjected into Xenopus oocytes.Northern blot analysis of poly(A)⁺RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFCwas found to be fourfold higher than uptake in control sublines. Thisincrease in folic acid and 5-MTHF uptake was inhibited by treatingIEC-6/RFC cells with cholesterol-modified antisense DNAoligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity(V_max) of theuptake process [the apparent Michaelis-Menten constant(K_m) alsochanged (range was 0.31 to 1.56 µM), but no specific trend wasseen]. In both IEC-6/RFC and control sublines, the uptake of bothfolic acid and 5-MTHF displayed 1)pH dependency, with a higher uptake at acidic pH 5.5 compared with pH7.5, and 2) inhibition to the sameextent by both reduced and oxidized folate derivatives. Thesecharacteristics are very similar to those seen in native intestinalepithelial cells. In contrast, RFC expressed inXenopus oocytes showed1) higher uptake at neutral andalkaline pH 7.5 compared with acidic pH 5.5 and2) higher sensitivity to reducedcompared with oxidized folate derivatives. Results of these studiesdemonstrate that the characteristics of RFC vary depending on the cellsystem in which it is expressed. Furthermore, the results may suggestthe involvement of cell- or tissue-specific posttranslationalmodification(s) and/or the existence of an auxiliary proteinthat may account for the differences in the characteristics of theintestinal RFC when expressed inXenopus oocytes compared with whenexpressed in intestinal epithelial cells.