Apparent Uptake Research Articles

Effect of pH on Na(+)-dependent phosphate transport in renal outer cortical and outer medullary BBMV

The influence of pH on sodium-phosphate cotransport was determined in brush-border membrane vesicles (BBMV) isolated from outer cortical and outer medullary tissue of porcine kidneys. Two transport systems are apparent in outer cortical brush-border vesicles, and one process is apparent in outer medullary vesicles at all pH values. The apparent maximum uptake rate (Vmax) of the low-affinity system in outer cortex vesicles decreased from 8.3 +/- 1.7 to 3.2 +/- 0.05 nmol.mg protein-1.min-1 with pH change of 8.0 to 6.0, and the high-affinity process changed from 1.3 +/- 0.2 to 0.1 +/- 0.01 nmol.mg protein-1.min-1. The respective affinity values (Km) also decreased 5.5 +/- 0.9 to 0.6 +/- 0.01 mM and 0.08 +/- 0.005 to 0.01 +/- 0.005 mM, respectively, with acidification. In outer medullary vesicles a decrease in pH diminished the apparent Km, 0.28 +/- 0.03 to 0.02 +/- 0.003 mM, and mean Vmax from 3.0 +/- 0.07 to 0.5 +/- 0.1 nmol.mg protein-1.min-1. The mean KNaD values were 22.1 +/- 4.2 mM in outer cortical vesicles (low-affinity system) and 58.7 +/- 7.2 mM in outer medullary vesicles (high-affinity system) and were not altered by pH, suggesting that H+ does not affect the sodium interactive site. The data suggest that the vesicles prepared from outer cortical and outer medullary tissue possess distinctive sodium-phosphate transporters that are sensitive to external H+ concentrations.

Scintigraphic Study of Radiolabelled Interferon-?? in Osteosarcoma Patients

Interferon-alpha is currently under evaluation as an antineoplastic agent in several types of tumour. Despite its clear in vitro effects, the effectiveness of interferon in vivo is limited. To assess whether this discrepancy reflects pharmacokinetic limitations, the authors analysed interferon distribution in 2 osteosarcoma patients by scintigraphy using 123I-interferon-alpha-2a. Numerical analysis of the scintigraphic records demonstrated that the main organs of elimination were the kidneys, when the calculation was made on the basis of surface area. On the other hand, the apparent total uptake by liver (whose projection surface--i.e. the area exposed to the lens--is greater) was higher, reaching about 25 to 30% of the injected dose. The projection surface of the tumour was able to take up radiolabelled interferon in both cases, resulting in a 4-fold increase in the external radiation count compared with the equivalent region of the contralateral limb (although it is not possible to determine whether the label is present on the tumour itself or on the surrounding inflammatory cells). Thus, interferon-alpha seems able to reach at least the immediate neighbourhood of osteosarcoma mass.

Dysfunction of choline transport system through blood-brain barrier in stroke-prone spontaneously hypertensive rats.

The blood-brain barrier (BBB) transport of choline was compared between stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar KY rats (WKY). The permeability surface area product (PS) of [3H]choline through the BBB in SHRSP (3.03 X 10(-3) +/- 1.09 X 10(-3) ml/min/g brain) was significantly lower than that in WKY (7.23 X 10(-3) +/- 0.97 X 10(-3) ml/min/g brain) in the presence of respective rat sera. No significant difference in the brain vascular space was indicated from the apparent uptake of [3H]sucrose between SHRSP and SKY. There was no significant difference for the Michaelis constant of choline transport between SHRSP (262 +/- 97 microM) and WKY (180 +/- 32 microM). However, the maximum velocity in SHRSP (3.41 +/- 1.19 nmol/min/g brain) was 37% lower than in WKY (5.40 +/- 0.38 nmol/min/g brain). Brain microdialysis technique was employed to collect the brain interstitial fluid in the rat hippocampus. The concentration of free choline in the brain dialysate in SHRSP was about half of that in WKY, while no significant difference was observed for the plasma concentration of free choline between SHRSP and WKY. In contrast, no significant difference was observed for the transport of D-[3H]glucose, 3-methyl-[3H]D-glucose and [3H]-phenylalanine through the BBB between SHRSP and WKY. Accordingly, the decreased choline concentration in the brain interstitial fluid ascribed to the specific dysfunction of the BBB choline transport has been demonstrated in SHRSP.

Hexose Accumulation and Turgor-Sensitive Starch Synthesis in Discs Derived from Source versus Sink Potato Tubers

The net total uptake (sum of soluble and insoluble components) of the hexoses, D-glucose and D-fructose, into sink potato (Solanum tuberosum L.) storage parenchyma was biphasic with respect to substrate concentration. Analysis of radioactive products revealed that the biphasic kinetics were composed of a linear, soluble component superimposed on saturating starch synthesis. In contrast, in source tuber tissue, there was negligible conversion of D-glucose to starch and the shape of the kinetic was the result of a biphasic soluble component. The uptake of D-fructose into source tissue was linear with respect to substrate concentration. Uptake of the non-metabolizable glucose analogue, 3-oxymethyl-D-glucose (3-OMG), into both sink and source tissue, demonstrated biphasic kinetics, indicating the presence of a carrier for glucose. The data demonstrate that in sink potato tubers, metabolism greatly influences apparent uptake kinetics, the kinetics of starch synthesis masking the kinetics of hexose transport at the plasmalemma. Uptake of L-glucose was linear with respect to substrate concentration, an observation consistent with this sugar not being recognized by a carrier. As in the case of sucrose, in sink tuber tissue the conversion of D-glucose and D-fructose to starch was sensitive to turgor, showing a marked optimum in external osmotica containing 300 mol m−3 mannitol. The mechanisms controlling turgor-sensitive starch synthesis in the potato tuber would, therefore, appear to be common to all three sugars.

Effect of dietary cholesterol on phosphatidylcholines and phosphatidylethanolamines in bile and gallbladder mucosa in the prairie dog

Humans with cholesterol gallstones have been reported to have alterations in the molecular species of phospholipids in bile. Both decreases in phospholipids with linoleic acid and increases in those with arachidonic acid have been found. The purpose of this study was to investigate the effect of a lithogenic diet (0.34% cholesterol) on the relative abundance of individual molecular species of phospholipids in the biliary tract of the prairie dog. In hepatic bile, cholesterol feeding resulted in increases in phospholipid species containing arachidonate and decreases in the major species containing its precursor, linoleate. In gallbladder bile of both control and cholesterol-fed animals, phospholipid species containing linoleate were significantly less abundant than in hepatic bile, suggesting that linoleoyl species were selectively absorbed by the gallbladder epithelium. This apparent uptake was significantly increased by cholesterol feeding. Phosphatidylcholines and phosphatidylethanolamines containing arachidonate were also significantly increased in the gallbladder mucosa of the cholesterol-fed animals. These increases in arachidonate-containing phospholipids in the gallbladder mucosa may contribute to the increase in gallbladder prostaglandin synthesis that precedes gallstone formation in this animal model.

Use of fluorescent dye BCECF to measure intracellular pH in cortical collecting tubule.

The compound 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) is being extensively used to measure intracellular pH (pHi) in a variety of tissue preparations. Use of this dye in the isolated perfused rabbit cortical collecting tubule (CCT) revealed two previously unreported findings. Apical incubation via luminal perfusion with the acetoxymethyl ester of BCECF (BCECF-AM) resulted in intense uptake into a minority cell population. Basolateral incubation via the peritubular bathing solution resulted in apparent homogenous uptake into all cells. The minority cells were identified as intercalated cells by the use of fluorescent probes specific for intercalated cells. Minority cell pHi was 7.34 +/- 0.06 (n = 9); majority cell pHi was 7.41 +/- 0.05 (n = 6). In addition, prolonged excitation of BCECF resulted in adverse effects. Intense continuous excitation for 1-3 min resulted in a fall in apparent pHi of 0.53 +/- 0.05 pH units (n = 6, P less than 0.001). After reloading with fresh BCECF-AM, the calculated pHi was 0.05 +/- 0.07 pH units higher than before prolonged excitation (P = NS), suggesting that the apparent decrease was due to changes in the pH sensitivity of BCECF as a result of the prolonged excitation. Prolonged excitation of cells with pHi clamped at 7.50 resulted in a decline in apparent pHi at the rate of 0.15 +/- 0.02 pH units/min (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

A soil test based n recommendation model for dryland wheat

Abstract Since soil test based N recommendations are not practiced in Morocco, this study was conducted to develop a N recommendation model based on soil nitrate level and other parameters for dryland wheat (Triticum aestivum L.) grown in common rotations in shallow (Rendolls), moderately deep (Calcixerolls) and deep (Chromoxererts) soils of Settat Province in Morocco. Sixteen N‐P Factorial experiments were conducted. Significant grain yield increases due to N fertilization averaged 76% over the check plot yields in all sites and in both seasons. Uptake of N was highly related to soil nitrates only in deeper soils with r2 values of 0.89, 0.76, and 0.64 for 20‐, 40‐ and 60‐cm deep profiles. Apparent uptake efficiency of fertilizer N averaged 34% in the drier year and 50% in the wet year. Relative yield was a linear function of initial soil nitrate content in 20‐, 40‐ and 60‐cm profiles with r2 values of 0.87, 0.94, and 0.89, respectively. For maximum grain yield 18, 31, and 50 kg of nitrate N/ha were required in 20‐, 40‐ and 60‐cm profiles, respectively. Nitrogen mineralization potentials were 144, 164, and 179 mg/kg for the above soils, respectively. A model based on critical level of soil nitrates, initial level of soil nitrates and apparent uptake efficiencies of soil and fertilizer N was developed to predict the fertilizer N requirement of wheat in deeper soils. If legume or mixed volunteer pastures preceded the wheat crop, the N fertilizer requirement was lower.

Cholinergic properties of rat atrial subcellular preparations

Several presynaptic cholinergic markers were measured in subcellular fractions of the rat cardiac atrium. A P 2 fraction consisting of isolated terminals, free mitochondria, glycogen and other subcellular organelles was prepared first by sucrose density centrifugation. No intact cells were recovered in this fraction, as determined by electron microscopy. Hemicholinium-3 sensitive choline transport coupled to acetylcholine synthesis was concentrated over 10-fold in the P 2 fraction compared to minced atria when expressed per mg protein. Six subfractions were recovered after centrifugation of the P 2 fraction over a sucrose-metrizamide density gradient. One of these (fraction 4) consistently contained higher levels of choline acetyltransferase activity and hemicholinium-3 sensitive [ 3H]acetylcholine synthesis than were present in the P 2 fraction of the other five subfractions. Electron microscopy of fraction 4 revealed isolated nerve terminals among other subcellular organelles. Kinetic analysis of total [ 3H]choline uptake in the P 2 fraction revealed two apparent uptake processes, with K t s of approximately 11 μ m and 219 μ m. [ 3H]Acetylcholine synthesis was partially sodium-dependent in the P 2 fraction, and reached a maximal level between choline concentrations of 100 to 200 μ m. Some of the newly synthesized [ 3H]-acetylcholine in the P 2 fraction was released by 50 m m K + depolarization in a calcium-dependent manner, arguing for a neuronal localization. This depolarization-induced release was attenuated by 10 to 100 μ m oxotremorine in an atropine-sensitive manner, but was not affected by 1 μ m tetrodotoxin.

The effect of dissolved organic matter on the bioavailability of polychlorinated dibenzo- p-dioxins

The extent to which dissolved organic matter (DOM) influences the bioavailability of polychlorinated dioxins (PCDDs) was determined using Aldrich humic acid or filtered natural lake water as sources of DOM. The bioconcentration factors (BCFs) for rainbow trout ( Salmo gairdneri) of four PCDD congeners were determined using both centrifuged and reverse-phase cartridge extractable water concentrations. Aldrich humic acid (DOM) reduced the apparent uptake rate constants of PCDDs by reducing the free water concentrations available to the fish. However, DOM from natural lake water had little effect on the apparent uptake rate constants relative to Aldrich humic acid. Although Aldrich humic acid overestimated the influence of DOM on BCFs relative to natural lake waters it did provide a useful tool to examine these interactions. Measured apparent BCFs of PCDDs were below those predicted by their octanol to water partition coefficients ( K ow) alone but only part of this reduction was explained by calculating the true BCFs using the estimated free water concentrations. Although this may be due to experimental errors leading to an overestimation of the free water concentrations, steric hindrances, solubility factors, or metabolic transformation may also be important factors controlling the BCFs of PCDDs.

Phospholipase Activation and Arachidonic Acid Release in Cultured Intestinal Epithelial Cells (INT 407)

The release of free arachidonic acid (AA) in cultured intestinal epithelial cells (INT 407) was investigated. INT-407 cells were first incubated overnight with radiolabeled 14C-AA, and most of the incorporated 14C-AA esterified into phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol. Labeled cells were then exposed to different stimulating agents and the release of free 14C-AA determined. The calcium ionophore A23187 caused a dose-dependent AA release that was preceded by a rapid uptake and a subsequent efflux of 45Ca2+. By contrast, phospholipase C from Clostridium perfringens caused a great AA release that was accompanied by an apparent uptake and a sustained intracellular accumulation of 45Ca2+. The cells alos released AA when exposed to the protein kinase C activator, 4 beta-phorbol-12-myristate-13-acetate (PMA), and this agent, like the diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol, significantly potentiated the AA release caused by A23187. Not only A23187-mediated but also phospholipase C- and PMA-mediated AA release was inhibited by 4-bromophenacyl bromide, a known phospholipase A2 inhibitor. These findings, taken together, indicate that AA release in intestinal epithelial cells can be caused by (i) Ca2+-mediated phospholipase activation, (ii) products of phospholipase C activity, and (iii) stimulation of protein kinase C. It is suggested, therefore, that AA release in intestinal epithelial cells is governed by intracellular Ca2+, protein kinase C-mediated protein phosphorylation, and activation of phospholipase A2.

Apparent inosine uptake by the human heart.

Although inosine has been used clinically to support the myocardium, no data are available on the fate of exogenous inosine in the human heart. We therefore infused six patients, catheterised for coronary angiography, with inosine (5 mg.kg-1.min-1 intravenously) for 6 minutes. Before infusion, the arterio-venous difference of inosine, hypoxanthine and xanthine across the heart was nil. During infusion, arterial inosine increased substantially, exceeding the coronary sinus concentration by a maximum of 200 (SEM 53) mumol.litre-1, p = 0.02, at the fourth minute. Arterial hypoxanthine and xanthine also increased, while the arterio-venous difference became 16(11) and 10(3) (p = 0.04) mumol.litre-1, respectively. Left ventricular dP/dtmax increased by 22(7)% (p = 0.04) at the end of infusion. Thus, there seemed to be substantial uptake of inosine by the human heart, followed by improvement in haemodynamics.

Effects of lime on the residual effectiveness of phosphate in acid soils

The effectiveness of phosphate, applied 9 months previously, was compared with that of freshly applied phosphate in relation to lime-induced changes in several phosphate characteristics of 14 phosphate-deficient, acidic soils. Effectiveness was measured in terms of the yield and phosphorus uptake of white clover herbage in a glasshouse experiment. Lime increased yields on the majority of soils, whether phosphate was applied or not. These were accompanied by increases in uptake of soil phosphate, which were usually associated with higher levels of exchangeable and soluble phosphate but not of sodium bicarbonate-extractable phosphate. On the other hand, lime had much more variable effects on the apparent uptake of fertilizer phosphate which were unrelated to lime-induced changes in soil phosphate sorptivity. Lime increased the uptake of previously applied phosphate in all but two soils, and it had a more beneficial or less detrimental effect on the uptake of old phosphate than of fresh phosphate in 9 of the 14 soils. Because lime usually increases the uptake of soil phosphate but often depresses the uptake and effectiveness of fertilizer phosphate, the more beneficial effect of lime on phosphate residues is consistent with their partial transformation into compositions resembling native soil phosphates.

Stimulation of calcium uptake by norepinephrine or high external potassium in human calyces and renal pelvis

The effects of stimulation with either 10 mumol/l norepinephrine or 85 mmol/l extracellular potassium concentration on calcium uptake were studied in muscle strips from human renal calyces and from the renal pelvis. The apparent uptake of calcium under control conditions was essentially complete after 30 min. Stimulation of the muscle strips with norepinephrine or high external potassium significantly (P less than 0.05) increased the calcium uptake over the control values at 30 and 100 min, whereas 45Ca efflux was virtually not affected. It is concluded that the mechanical responses of the muscle strips to norepinephrine or high external potassium correspond with an increased uptake of calcium suggesting that both types of activation are mediated by an increase of intracellular calcium concentration similar to that described in other smooth muscles.

Calcium Antagonist TMB-8 Inhibits Cell Wall Formation and Growth in Pea

The effects on auxin-stimulated growth and cell wall formation of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate.HCl (TMB-8), an intracellular Ca2+ antagonist, were investigated in abraded stem segments from aetiolated seedlings of Pisum sativum L. cv. Alaska. Incubation of segments at pH 6-0 with 200 mmol m~3 TMB-8 resulted in a 50% inhibition of auxin-stimulated growth. Added Ca2 + did not restore normal auxin-stimulated growth, presumably because of its well-known stiffening effect on the cell wall. In segments incubated at a pH (7-2) which prevented elongation, auxin promoted the incorporation of [3H]glucose into the cell wall relative to total uptake of label. TMB-8 abolished about 60% of the total incorporation of label into cell walls in the presence of auxin, but was not effective in the absence of auxin. Exogenous CaCl2 reversed the inhibitory effect of TMB-8 on relative cell wall incorporation in a parabolic manner, with a 50% reversal at about 100 mmol m~3 and complete reversal at 1-0 mol m-3 Ca2 + . Other ions tested (Mg2+, Mn2+, Cu2 + , Zn2+) were without substantial effect at concentrations of 0-5 mol rn3. Both apparent uptake of Ca2+ and consequent reversal of TMB-8 inhibition of cell wall incorporation were blocked by the Ca2+ channel blockers verapamil and La3 + . The data provide further evidence that auxin-stimulated growth is dependent upon continued cell wall incorporation, and suggest that a Ca2 + messenger system may be involved in the promotory actions of auxin on cell wall synthesis and long-term growth.

Inhibition of Uptake of Thyroid Hormone into Rat Hepatocytes by Preincubation withN-Bromoacetyl 3,3′,5-Triiodothy r onine

To investigate whether affinity coupling of N-bromoacetyl-T3 (BrAcT3) to the T3 membrane carrier results in an inhibition of transport of T3 into the cell, rat hepatocytes in monolayer were incubated for 2 h at 21 C with 1.3 mumol/liter BrAcT3 in medium without protein. After extensive washing, cells were incubated during 20 h at 37 C with [125I]T3 in medium with 0.5% BSA, and products in supernatants were analyzed by LH-20 column chromatography. In addition the apparent affinity constant (Km) and maximal uptake velocity (Vmax) of the high affinity uptake process were estimated using 1 min incubations of hepatocytes with various concentrations of T3. In control experiments (i.e. without BrAcT3 affinity coupling) about 57% of the added T3 was cleared from the medium and further metabolized, 85% of the cleared T3 reappeared in the medium as I-, 15% as conjugates. Addition of propylthiouracil during the 20 h incubation with T3 strongly inhibited deiodination, without a change in T3 clearance. Because T3 is sulfated before deiodination, a concomitant rise in conjugates was observed. Addition of ouabain to control cells during the 20 h incubation with T3 strongly inhibited uptake, with a parallel decrease in I- and conjugate formation. After affinity coupling of BrAcT3, T3 clearance was inhibited (by 30% P less than 0.001). Since I- production was more depressed (by 73%) than T3 clearance, with some rise in conjugate formation (P less than 0.001), inhibition of deiodinase by BrAcT3 also took place. The effects of BrAcT3 and ouabain on uptake of T3 appeared to be additive as were the effects of propylthiouracil and BrAcT3 on deiodination. After affinity coupling of BrAcT3, the Km of T3 uptake did not change significantly; however Vmax was 54% lower (P less than 0.025) indicating a noncompetitive inhibition of the transport system. Preincubation of the cells with N-acetyl-T3 does not alter the characteristics of uptake of T3 by rat hepatocytes as compared to controls, indicating that no binding of this compound occurs. It is concluded that preincubation of hepatocytes with BrAcT3 diminished I- formation from T3; 50% of this inhibition is due to decreased membrane transport and 50% by reduction of deiodination. Inhibition of membrane transport by BrAcT3 is substantiated by a 54% lower Vmax without a significant change in Km as compared to control. The effect of transport of thyroid hormone on metabolism stresses the importance of the membrane carrier in the translocation process.

Platelet serotonin uptake: Methods and strain differences

The pharmacokinetic properties of platelets make these vascular elements a potential and convenient model for serotoninergic neurons. Because we encountered several difficulties in preliminary platelet serotonin (5HT) uptake assays, we explored the influence of several assay factors on the parameters obtained. We found that a citrate-dextrose anticoagulant stabilized platelet 5HT uptake even at room temperature. More rapid wash rates during the filtration following the assay incubation gave higher apparent uptake affinities. Platelets from an inbred strain of rats exhibited less variability in V max than cells from outbred strains studied. These findings, in conjunction with a human twin study also suggesting that variance in platelet 5HT uptake increases with genetic diversification, demonstrate some additional confounding factors in platelet 5HT uptake studies.

Modifying effects of pH and temperature on ( 14C)erythromycin uptake into Staphylococcus aureus — Relation to antimicrobial activity

The uptake of (14C)erythromycin into Staphylococcus aureus was investigated by use of a rapid centrifugation method. Erythromycin uptake was saturable with time and with increasing erythromycin concentrations (apparent uptake constant Km = 6.0 x 10(-7) moles/l). Inhibitors of glycolysis, respiration and oxidative phosphorylation did not influence the uptake process but uptake was decreased by reducing temperature. Increases of erythromycin uptake, decreases of half life times of the uptake reaction and a log dose linked to enhancement of antimicrobial activity were seen with alkaline pH levels of the incubation medium. The experimental data conform well with the concept of non ionic diffusion. The high affinity of erythromycin to the intracellular ribosomal target site probably generates the driving force of uptake and the unionized antibiotic obviously represents the antimicrobially active molecular form.

Chloramphenicol accumulation by Haemophilus influenzae.

The mechanism of chloramphenicol transport into susceptible strains of Haemophilus influenzae cells has not been reported previously. We examined apparent uptake of chloramphenicol by bacterial cells by using high-pressure liquid chromatography to quantitate drug disappearance from liquid media. Cell-associated chloramphenicol concentration is 1,000-fold greater than the extracellular drug concentration. Under incubation conditions associated with chloramphenicol disappearance from media, cellular protein synthesis was inhibited; however, if accumulation was inhibited, protein synthesis occurred in the presence of the drug. Chloramphenicol uptake appeared saturable (Km = 0.96 mM, Vmax = 0.9 mumol/min per mg of protein) and energy dependent: disappearance from media was markedly decreased by 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, compounds which disrupt the proton motive force. Uptake occurred only in median which can support growth and was dependent upon temperature and pH. Drug accumulation was minimally affected by inhibitors of electron transport or by gentamicin and puromycin, both inhibitors of protein synthesis. The rate of disappearance was inhibited by SCH24893, a fluorinated chloramphenicol analog which also inhibits protein synthesis. We conclude that chloramphenicol accumulation by H. influenzae occurs by energy-dependent transport.

Removal of Hazardous Organic Pollutants by Adsorption on Microbial Biomass

Removal of five toxic organic compounds from water by adsorption on microbial biomass was investigated. Lindane, diazinon, malathion, pentachlorophenol, and the PCB 2-chlorobiphenyl were adsorbed onto two types of inactive microbial biomass (a pure strain of Rhizopusarrhizus, and a mixed culture of activated sludge). Desorption and the thermodynamics of the adsorption process were also investigated. With the exception of malathion, the adsorptive uptake data fit the Freundlich equation and were well correlated with the octanol/water partition coefficient, but not as well correlated with water solubility of the compounds. Except for malathion the adsorption was reversible. Malathion showed unusually high apparent uptake and the removal appeared to be irreversible. The experimental evidence suggests that the disappearance of malathion resulted from a chemical reaction, while physical adsorption appears to account for removal of the other compounds.

Apparent inosine uptake by the human heart

Apparent inosine uptake by the human heart