Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

Uptake and intracellular fate of L-DOPA in a human intestinal epithelial cell line: Caco-2

The aim of the presentstudy was to examine the kinetic characteristics of theL-3,4-dihydroxyphenylalanine (L-DOPA)transporter and the fate of newly formed dopamine in Caco-2 cells. Inthe presence of 50 µM benserazide (an inhibitor of aromaticL-amino acid decarboxylase), L-DOPA was rapidlyaccumulated in Caco-2 cells. At equilibrium (30 min of incubation) theintracellular L-DOPA concentration was 10.2 ± 0.1 µM ata medium concentration of 0.5 µM. In saturation experiments theaccumulation of L-DOPA was saturable with aMichaelis-Menten constant (K_m) of 60 ± 10 µMand a maximal reaction velocity (V_max) of 6.6 ± 0.3 nmol · mg protein¹ · 6 min¹; at 4°C the amount of L-DOPAaccumulated in the cells was nonsaturable. When cells were incubatedwith increasing concentrations of L-DOPA (10-100 µM)in the absence of benserazide, a substantial amount of theL-DOPA that was taken up was decarboxylated to dopamine, with an apparent K_m of 27.2 µM. In experimentsperformed in cells cultured in polycarbonate filters, theaccumulation of L-DOPA in the presence of benserazide wasgreater when the substrate was applied from the basolateral cell borderthan when it was applied from the apical cell border. In the absence ofbenserazide, L-DOPA applied from the basolateral cellborder resulted in a nonlinear formation of dopamine(K_m = 43 ± 7 µM,V_max = 23.7 ± 1.2 nmol · mgprotein¹ · 6 min¹). Theamount of dopamine leaving the cell through the apical cell border waslower than the amount that escaped through the basolateral cell border,and the process was saturable (K_m = 623 ± 238 µM, V_max = 0.19 ± 0.02 nmol · mgprotein¹ · 6 min¹). Inconclusion, the data presented here show that Caco-2 cells are endowedwith an efficient L-DOPA uptake system, and intracellular L-DOPA was found to be rapidly converted to dopamine, someof which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way tolook at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.

     

Contraction-induced increase in Vmax of palmitate uptake and oxidation in perfused skeletal muscle

To evaluate the effects of contractions on thekinetics of uptake and oxidation of palmitate in a physiological musclepreparation, rat hindquarters were perfused with glucose (6 mmol/l),albumin-bound [1-¹⁴C]palmitate, andvarying amounts of albumin-bound palmitate (200-2,200 µmol/l) atrest and during muscle contractions. When plotted against the unboundpalmitate concentration, palmitate uptake and oxidation displayedsimple Michaelis-Menten kinetics with estimated maximal velocity(V_max)and Michaelis-Menten constant(K_m) values of42.8 ± 3.8 (SE)nmol · min¹ · g¹and 13.4 ± 3.4 nmol/l for palmitate uptake and 3.8 ± 0.4 nmol · min¹ · g¹and 8.1 ± 2.9 nmol/l for palmitate oxidation, respectively, at rest.Whereas muscle contractions increased theV_maxfor both palmitate uptake and oxidation to 91.6 ± 10.1 and 16.5 ± 2.3 nmol · min¹ · g¹,respectively, theK_m remainedunchanged.V_maxand K_m estimates obtained from Hanes-Woolf plots (substrate concentration/velocity vs.substrate concentration) were not significantly different. In theresting perfused hindquarter, an increase in palmitate delivery from31.9 ± 0.9 to 48.7 ± 1.2 µmol · g¹ · h¹by increasing perfusate flow was associated with a decrease in thefractional uptake of palmitate so that the rates of uptake andoxidation of palmitate remained unchanged. It is concluded that therates of uptake and oxidation of long-chain fatty acids (LCFA) saturatewith an increase in the concentration of unbound LCFA in perfusedskeletal muscle and that muscle contractions, but not an increase inplasma flow, increase theV_maxfor LCFA uptake and oxidation. The data are consistent with the notion that uptake of LCFA in muscle may be mediated in part by a transport system.

     

Characterization of L-glutamine transport by a human neuroblastoma cell line

This study characterized theNa⁺-dependent transport of L-glutamine by ahuman neuroblastoma cell line, SK-N-SH. The Na⁺-dependentcomponent represented >95% of the total glutamine uptake. Kineticstudies showed a single saturable high-affinity carrier with aMichaelis constant (K_m) of 163 ± 23 µMand a maximum transport velocity (V_max) of13,713 ± 803 pmol · mgprotein¹ · min¹. Glutamine uptakewas markedly inhibited in the presence of L-alanine, L-asparagine, and L-serine. Li⁺ didnot substitute for Na⁺. These data show thatL-glutamine is predominantly taken up through systemASC. Glutamine deprivation resulted in the decrease of glutamine transport by a mechanism that decreasedV_max without affectingK_m. The expression of the system ASC subtypeASCT2 decreased in the glutamine-deprived group, whereas glutaminedeprivation did not induce changes in system ASC subtype ASCT1 mRNAexpression. Adaptive increases in Na⁺-dependent glutamate,Na⁺-dependent 2-(methylamino)isobutyric acid, andNa⁺-independent leucine transport were observed underglutamine-deprived conditions, which were completely blocked byactinomycin D and cycloheximide. These mechanisms may allow cells tosurvive and even grow under nutrient-deprived conditions.

     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

Riboflavin uptake by human-derived colonic epithelial NCM460 cells

Normal microflora ofthe large intestine synthesize a number of water-soluble vitaminsincluding riboflavin (RF). Recent studies have shown that colonicepithelial cells posses an efficient carrier-mediated mechanism forabsorbing some of these micronutrients. The aim of the present studywas to determine whether colonic cells also posses a carrier-mediatedmechanism for RF uptake and, if so, to characterize this mechanism andstudy its cellular regulation. Confluent monolayers of thehuman-derived nontransformed colonic epithelial cells NCM460 and[³H]RF were used in the study. Uptake of RF wasfound to be 1) appreciable and temperature and energydependent; 2) Na⁺ independent; 3) saturableas a function of concentration with an apparent K_mof 0.14 µM and V_max of 3.29 pmol · mgprotein¹ · 3 min¹; 4) inhibited by the structural analogslumiflavin and lumichrome (K_i of 1.8 and 14.1 µM,respectively) but not by the unrelated biotin; 5) inhibited ina competitive manner by the membrane transport inhibitor amiloride(K_i = 0.86 mM) but not by furosemide, DIDS, orprobenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RFuptake in RF-deficient and oversupplemented conditions, respectively;and 7) modulated by an intracellularCa²⁺/calmodulin-mediated pathway. These studies demonstratefor the first time the existence of a specialized carrier-mediatedmechanism for RF uptake in an in vitro cellular model system of humancolonocytes. This mechanism appears to be regulated by extracellularsubstrate level and by an intracellularCa²⁺/calmodulin-mediated pathway. It is suggested that theidentified transport system may be involved in the absorption ofbacterially synthesized RF in the large intestine and that this sourceof RF may contribute toward RF homeostasis, especially that of colonocytes.

     

Sodium-Stimulation of Phosphate Uptake in the Cyanobacterium Anabaena PCC 7119

Anabaena PCC 7119 showed higher rates of phosphate uptake whencells were under P-starvation. Phosphate uptake was energy-dependentas indicated the decrease observed when assays were performedin the dark or in the presence of inhibitors of photosyntheticelectron transport, energy transfer and adenosine triphosphataseactivity. Phosphate uptake was stimulated by Na⁺ both in P-sufficientcells and P-starved cells. Li⁺ and K⁺ acted as partial analoguesfor Na⁺. The Na⁺-stimulation of phosphate uptake followed Michaelis-Mentenkinetics, half-saturation (K) of phosphate uptake was reachedwith a Na⁺ concentration of 212 µM. The absence of Na⁺reduced the rates of phosphate uptake at all phosphate concentrationsassayed (1–20 µM). The maximum uptake rates (V_max)decreased from 658 nmol P (mg dry wt)^-1 h^-1 in the presenceof Na⁺ to 149 nmol P (mg dry wt)^-1 h^-1 in the absence of Na⁺.The absence of Na⁺ did not change significantly the concentrationof phosphate required to reach half-saturation (K) (3.01 µMin the presence of Na⁺ vs 3.21 µM in the absence of Na⁺).In the presence of Na⁺ the rate of phosphate uptake was affectedby the pH; optimal rates were observed at pH 8. In the absenceof Na⁺ phosphate uptake was not affected by the pH; low rateswere observed in all cases. Monensin, an ionophore which collapsesNa⁺-gradients, reduced the rate of phosphate uptake in Na⁺-supplementedcells. These results indicated the existence of a Na⁺-dependentphosphate uptake in Anabaena PCC 7119. (Received September 8, 1992; Accepted November 17, 1992)     

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.

     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

L-Carnitine transport in human placental brush-border membranes is mediated by the sodium-dependent organic cation transporter OCTN2

Maternofetal transport of L-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that L-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K_m = 11.09 ± 1.32 µM; V_max = 41.75 ± 0.94 pmol·mg protein^–1·min^–1), and was unchanged over the pH range from 5.5 to 8.5. L-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of L-carnitine, including D-carnitine, acetyl-D,L-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-L-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na⁺-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for L-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans. membrane transport; valproate; maternofetal; xenobiotics; acylcarnitine     

Uptake of Inorganic Phosphate by Suspension-Cultured Tobacco Cells: Kinetics and Regulation by Pi Starvation

The kinetics of P_i uptake by phosphate-starved and non-starvedtobacco cells (Nicotiana tabacum BY-2) suspension culture wasinvestigated. The kinetic parameters of P_i uptake were determinedby computer simulation of the curve that represented the time-dependentloss of P_i from the culture medium. The uptake profile couldbe completely explained by assuming the existence of only onekind of Michaelis-Menten-type P_i-transport system with an affinityfor P_i (K_m) of about 2.5 µM (the lowest value reportedto date) in both P_i-starved and non-starved cells. No evidencewas obtained suggesting the existence of a "low-affinity" P_i-uptakesystem that has been postulated to exist in several other plantmaterials. The V_max for uptake of P_i by non-starved cells was12 nmol per minute per milliliter of packed cell. Phosphatestarvation increased the V_max more than 5-fold, while it hadno effect on the affinity for P_i. V_max began to increase (atan almost constant rate) just after loss of all P_i from theculture medium and it reached a maximum about 16 hours later.This induction process was completely prevented by the additionof cycloheximide to the culture medium. All these results suggestthat P_i starvation increases the synthesis of a phosphate-carriercomplex that is postulated to be involved in the P_i-uptake process. (Received August 12, 1994; Accepted December 26, 1994)     

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells

The reabsorption of filtered di- andtripeptides as well as certain peptide mimetics from the tubular lumeninto renal epithelial cells is mediated by anH⁺-coupledhigh-affinity transport process. Here we demonstrate for the first timeH⁺-coupled uptake of dipeptidesinto the renal proximal tubule cell lineLLC-PK₁. Transport was assessed1) by uptake studies using theradiolabeled dipeptideD-[³H]Phe-L-Ala,2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and3) by measurement of intracellularpH (pH_i) changes as aconsequence of H⁺-coupleddipeptide transport. Uptake ofD-Phe-L-Alaincreased linearly over 11 days postconfluency and showed all thecharacteristics of the kidney cortex high-affinity peptide transporter,e.g., a pH optimum for transport ofD-Phe-L-Alaof 6.0, an apparent K_m value forinflux of 25.8 ± 3.6 µM, and affinities of differently chargeddipeptides or the -lactam antibiotic cefadroxil to the binding sitein the range of 20-80 µM.pH_i measurements established thepeptide transporter to induce pronounced intracellular acidification inLLC-PK₁ cells and confirm itspostulated role as a cellular acid loader.

     

Nitrate Uptake and Reduction of Aseptically Cultivated Spruce Seedlings, Picea abies (L.) Karst

Spruce (Picea abies (L.) Karst.) seedlings were asepticallycultivated and the effects of different N-nutrition on net uptakeand reduction of nitrate were investigated. The characteristicsof nitrate uptake were calculated, K_s as 0?2 mol m^–3 andV_max as 18 µmol g^–1 d^–1. Low pH, and Al³⁺ in the medium caused adecrease in nitrate uptake rate. An in vivo assay was set upwhich allowed the measurement of NRA in both roots and needlesof spruce seedlings. The in vivo nitrate reductase activitywas repressed by ammonium and stimulated by nitrate. Nitratereduction was similar to nitrate uptake, negatively affectedby low pH and ammonium. Therefore, a limited N-supply to spruceseemed to occur when pH was low in the rhizosphere combinedwith the presence of Al³⁺ and . Key words: Spruce, nitrate uptake, nitrate reduction     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

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     

Role of Coccoliths in the Utilization of Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi: a Survey Using Intact Cells and Protoplasts

The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O₂ at various concentrationsof externally added NaHCO₃ were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO₂-utilization reaction was, therefore, considered to involvetwo processes with different values of K_m and V_max. From thekinetic analyses, K_m and V_max [µmoles O₂ (ml PCV)^–1h^–1] were deduced to be 92 µM and 76.3 for a "low-K_m"reaction and 4.1 mM and 252 for a "high-K_m" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of ¹⁴C-DIC and the incorporation of ¹⁴C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO₂-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of ¹⁴C into CaCO₃ apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of ¹⁴C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of ¹⁴C incorporated into CaCO₃ was much smallerin the former than the latter. The production of Ca¹⁴CO₃ increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca¹⁴CCO₃ started to decrease. The decrease wasaccompanied by an increase in ¹⁴C in the products of photosynthesis,suggesting that CaCO₃ was reutilized for the photosyntheticfixation of CO₂ and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995)     

Pacemaker potentials generated by interstitial cells of Cajal in the murine intestine

Pacemaker potentials were recorded in situ from myenteric interstitial cells of Cajal (ICC-MY) in the murine small intestine. The nature of the two components of pacemaker potentials (upstroke and plateau) were investigated and compared with slow waves recorded from circular muscle cells. Pacemaker potentials and slow waves were not blocked by nifedipine (3 µM). In the presence of nifedipine, mibefradil, a voltage-dependent Ca²⁺ channel blocker, reduced the amplitude, frequency, and rate of rise of upstroke depolarization (dV/dt_max) of pacemaker potentials and slow waves in a dose-dependent manner (1–30 µM). Mibefradil (30 µM) changed the pattern of pacemaker potentials from rapidly rising, high-frequency events to slowly depolarizing, low-frequency events with considerable membrane noise (unitary potentials) between pacemaker potentials. Caffeine (3 mM) abolished pacemaker potentials in the presence of mibefradil. Pinacidil (10 µM), an ATP-sensitive K⁺ channel opener, hyperpolarized ICC-MY and increased the amplitude and dV/dt_max without affecting frequency. Pinacidil hyperpolarized smooth muscle cells and attenuated the amplitude and dV/dt_max of slow waves without affecting frequency. The effects of pinacidil were blocked by glibenclamide (10 µM). These data suggest that slow waves are electrotonic potentials driven by pacemaker potentials. The upstroke component of pacemaker potentials is due to activation of dihydropyridine-resistant Ca²⁺ channels, and this depolarization entrains pacemaker activity to create the plateau potential. The plateau potential may be due to summation of unitary potentials generated by individual or small groups of pacemaker units in ICC-MY. Entrainment of unitary potentials appears to depend on Ca²⁺ entry during upstroke depolarization. pacemaker activity; slow waves; gastrointestinal motility; calcium channel     

Comparative effects of a low-carbohydrate diet and exercise plus a low-carbohydrate diet on muscle sarcoplasmic reticulum responses in males

We employed a glycogen-depleting session of exercise followed by a low-carbohydrate (CHO) diet to investigate modifications that occur in muscle sarcoplasmic reticulum (SR) Ca²⁺-cycling properties compared with low-CHO diet alone. SR properties were assessed in nine untrained males [peak aerobic power (O_{2 peak}) = 43.6 ± 2.6 (SE) ml·kg^–1·min^–1] during prolonged cycle exercise to fatigue performed at 58% O_{2 peak} after 4 days of low-CHO diet (Lo CHO) and after glycogen-depleting exercise plus 4 days of low-CHO (Ex+Lo CHO). Compared with Lo CHO, Ex+Lo CHO resulted in 12% lower (P < 0.05) resting maximal Ca²⁺-ATPase activity (V_max = 174 ± 12 vs. 153 ± 10 µmol·g protein^–1·min^–1) and smaller reduction in V_max induced during exercise. A similar effect was observed for Ca²⁺ uptake. The Hill coefficient, defined as slope of the relationship between cytosolic free Ca²⁺ concentration and Ca²⁺-ATPase activity, was higher (P < 0.05) at rest (2.07 ± 0.15 vs. 1.90 ± 0.10) with Ex+Lo CHO, an effect that persisted throughout the exercise. The coupling ratio, defined as the ratio of Ca²⁺ uptake to V_max, was 23–30% elevated (P < 0.05) at rest and during the first 60 min of exercise with Ex+Lo CHO. The 27 and 34% reductions (P < 0.05) in phase 1 and phase 2 Ca²⁺ release, respectively, observed during exercise with Lo CHO were not altered by Ex+Lo CHO. These results indicate that when prolonged exercise precedes a short-term Lo CHO diet, Ca²⁺ sequestration properties and efficiency are improved compared with those during Lo CHO alone. calcium cycling; vastus lateralis; contractile activity; glycogen; phosphorylation potential     

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. K_m values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC₅₀ (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca²⁺/CaM complex (–55 ± 5%, n = 10 and –63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (–54 ± 5%, n = 14, and –31 ± 6%, n = 26) and PKA (–16 ± 5%, n = 16, and –18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca²⁺/CaM complex resulted in a significant decrease of V_max (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (–22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca²⁺/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride     

UPTAKE OF ACETATE BY NEOTRICULA APERTA (GASTROPODA: POMATIOPSIDAE), THE SNAIL HOST OF SCHISTOSOMA MEKONGI IN THE LOWER MEKONG BASIN

All three races of Neotricula aperta, an epilithic, schistosometransmitting, snail of the Mekong and Mul rivers of NortheastThailand and southern Laos, were found to take up acetate froma dilute solution. After 48 h incubation the mean specific netuptake rates (µmol^–1 g^–1 h^–1), from750 µM acetate, were: 1.86, -race; 1.39, ß-raceand 3.25, y-race. Over 48 h the snails were able to achievereductions in the ambient acetate concentration of up to 60%.Incubations under bacteriostatis suggested that bacteria arenot involved in the uptake of acetate by N. aperta. The uptakecharacteristics conform to the Michaelis-Menten model. The transportconstants, J_max (µmol^–1 g^–1 h^–1) andK_t (µM) were 1.10 and 102 respectively (-race). Racialdifferences in uptake characteristics are discussed in relationto micro-habitat differences. HPLC indicated concentrations of acetate in y-N. aperta microhabitatsof around 325 µM. This suggests a pool size sufficientto satisfy only 6% of the snail's basal metabolic rate (BMR).Levels within the epilithic aufwuchs, however, are probablyhigh enough to provide for more than 50% of the BMR. The possible role of acetate in the energy metabolism of N.aperta is discussed. Short-chain carboxylic acids (such as acetate),arising from the decomposition of the aufwuchs, could representsources of fermentable organics that may be taken up by N. apertasnails and used to supplement their nutrition during times offood shortage. However, further investigations involving ¹⁴C-labellingtechniques are required. The findings of this investigationhave implications for the chemical ecology and life-cycle ofN. aperta. (Received 16 June 1994; accepted 28 July 1994)