Stereospecificity at carbon 6 of fomyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

The unnatural diastereoisomer of l-5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [³H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K_i, were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K_i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.     

Choline and diethylcholine transport into a cholinergic clone of neuroblastoma cells.

The transport of choline and diethylcholine has been investigated in a cholinergic clone (S20F₃) of mouse neuroblastoma cells. Choline transport was linear for the first 20 min of incubation and was temperature dependent at low concentrations (< 1 × 10^?5 M). Diethylcholine transport was linear for the first 10 min of incubation and was also temperature dependent at low concentrations. High affinity (K_m < 1 × 10^?6 M) and low affinity (K_m > 1 × 10^?5 M) components of transport were found for both compounds. The transport system had a greater apparent affinity (lower K_m) for choline than for diethylcholine (3-fold), but maximal transport velocities were about equal. Each compound competitively inhibited the other's high affinity transport. Hemicholinium (1 × 10^?5 M) slightly inhibited high affinity choline transport but triethylcholine (1 × 10^?6?1 × 10^?4 M) did not. Choline transport was also found to be dependent on the pH and pCO₂ of the medium.     

Carrier-mediated transport of daunorubicin, adriamycin, and rubidazone in Ehrlich ascites tumour cells

The transport of DNR, ADR, and RBD was studied in Ehrlich ascites tumour cells in vitro. Within the first min intracellular drug binding, judged by measuring drug uptake in lysed cells considerably exceeded the uptake in whole cells, indicating that membrane transport is rate-limiting to drug uptake. Cellular net uptake showed a biphasic uptake pattern with a rapid phase within 5 sec and a subsequent linear phase of 1–4 min. The slope of the linear component was increased by sodium azide, suggesting an energy dependent efflux component for these drugs. To obtain near unidirectional influx the transport experiments were performed on cells inhibited by sodium azide. Several findings indicated a carrier mediated inward transport mechanism: (1) a high structural specificity for influx, (2) the influx exhibited simple saturation kinetics for each drug, (3) competition for transport was demonstrated by structural analogues, (4) an effect compatible with counter transport was demonstrated for DNR and (5) the influx of DNR was highly temperature-sensitive (Q₁₀ = 2.9). Considerable variation in both K_m and V_max was noted between the three derivatives. K_m and K_max were highest for DNR and lowest for ADR. Discrepancies between K_i and K_m indicated that ADR and RBD were less efficient inhibitors of [³H]DNR influx than would be expected from their K_m values. From the distribution of the drugs between chloroform/buffer and olive oil/buffer RBD appeared to be the most lipophilic drug; DNR proved nearly nine times more lipophilic than ADR.     

Anticonvulsant potencies of the enantiomers of the neurosteroids androsterone and etiocholanolone exceed those of the natural forms

Rationale

Androsterone [(3α,5α)-3-hydroxyandrostan-17-one; 5α,3α-A] and its 5β-epimer etiocholanolone [(3α,5β)-3-hydroxyandrostan-17-one; 5β,3α-A)], the major excreted metabolites of testosterone, are neurosteroid positive modulators of GABA_A receptors. Such neurosteroids typically show enantioselectivity in which the natural form is more potent than the corresponding unnatural enantiomer. For 5α,3α-A and 5β,3α-A, the unnatural enantiomers are more potent at GABA_A receptors than the natural forms.

Objectives

The aim of this study was to compare the anticonvulsant potencies and time courses of 5α,3α-A and 5β,3α-A with their enantiomers in mouse seizure models.

Methods

Steroids were administered intraperitoneally to male NIH Swiss mice 15 min (or up to 6 h in time course experiments) prior to administration of an electrical stimulus in the 6-Hz or maximal electroshock (MES) seizure tests or the convulsant pentylenetetrazol (PTZ).

Results

In the 6-Hz test, the ED₅₀ values of ent-5α,3α-A was 5.0 mg/kg whereas the value for 5α,3α-A was 12.1 mg/kg; the corresponding values in the PTZ seizure test were 22.8 and 51.8 mg/kg. Neurosteroid GABA_A receptor-positive allosteric modulators are generally weak in the MES seizure test and this was confirmed in the present study. However, the atypical relative potency relationship was maintained with ED₅₀ values of 140 and 223 mg/kg for ent-5α,3α-A and 5α,3α-A, respectively. Similar relationships were obtained for the 5β-isomers, except that the enantioselectivity was accentuated. In the 6-Hz and PTZ tests, the ED₅₀ values of ent-5β,3α-A were 11.8 and 20.4 mg/kg whereas the values for 5β,3α-A were 57.6 and 109.1 mg/kg. Protective activity in the 6-Hz test of ent-5α,3α-A persisted for somewhat longer (~5 h) than for 5α,3α-A (~4 h); protection by ent-5β,3α-A also persisted longer (~3 h) than for 5β,3α-A (~2 h).

Conclusions

The unnatural enantiomers of 17-keto androgen class neurosteroids have greater in vivo potency and a longer duration of action than their natural counterparts. The more prolonged duration of action of the unnatural enantiomers could reflect reduced susceptibility to metabolism. Unnatural enantiomers of androgen class neurosteroids could have therapeutic utility and may provide advantages over the corresponding natural isomers due to enhanced potency and improved pharmacokinetic characteristics.     

Characteristics of membrane transport of methotrexate by cultured human breast cancer cells

Methotrexate, a folic acid analogue, enters cells using a high-affinity carrier system that is shared with naturally occurring reduced folates. Methotrexate transport by MCF-7 cells, a hormonally responsive line of human breast cancer cells, exhibited a high-affinity carrier system that displayed Michaelis-Menten kinetics [K_m = 8.22 ± 0.62 μM; V_max = 12.22 ± 2.8 nmoles · min^?1·(g cell protein)^?1] was competitively inhibited by leucovorin and aminopterin, but not by folic acid; and was temperature-sensitive (Q₁₀ = 2.25 ± 0.32). Initial uptake rates were not affected by ouabain or sodium azide, but efflux of intracellular drug was inhibited markedly by sodium azide, suggesting the presence of an energy-dependent active efflux mechanism. At extracellular methotrexate concentrations in excess of 10 μM, a second, low-affinity uptake component could be identified that may represent a lower affinity membrane carrier or passive diffusion. Examination of hormonal influences on methotrexate transport revealed that growth of MCF-7 cells in serum-free medium induced a significant increase in the transport K_m value (15.93 ± 1.6 μM) compared to the K_m of 8.22 μM for cells grown in fetal calf serum. This change in affinity of the transport carrier could be reversed by the additon of insulin, but not of estradiol, to the culture medium. Methotrexate transport by human breast-cancer cells displayed characteristics that were similar to those of transport reported for human leukemia cells but that have not been documented previously for cells derived from a human solid tumor. In addition, the drug transport carrier was subject to modulation by insulin but not by estrogen.     

Histidine transport by isolated rat peritoneal mast cells

Kinetic constants for the transport of [³H]histidine into isolated rat peritoneal mast cells were determined. The value of K_m for histidine transport was 44.0 μM; the value of V_max under the same conditions was 18.9 pinoles · min^? · (10⁶ cells)^?1. These parameters did not change in value after the addition of exogenous histamine. The uptake of histidine and its decarboxylation to histamine were relatively rapid processes compared to the transfer of the newly formed histamine into mast cell granules, so that nascent histaramine appeared transiently in the cytoplasm. Amino-acid competition experiments support the assignment of L system transport for the bulk of histidine uptake by mast cells. Metabolic inhibitors that deplete cellular ATP did not inhibit the uptake process.     

Benzimidazolones enhance the function of epithelial Na+ transport

Background and Purpose

Pharmacological enhancement of vectorial Na⁺ transport may be useful to increase alveolar fluid clearance. Herein, we investigated the influence of the benzimidazolones 1-ethyl-1,3-dihydro-2-benzimidazolone (1-EBIO), 5,6-dichloro-1-EBIO (DC-EBIO) and chlorzoxazone on vectorial epithelial Na⁺ transport.

Experimental Approach

Effects on vectorial Na⁺ transport and amiloride-sensitive apical membrane Na⁺ permeability were determined by measuring short-circuit currents (I_SC) in rat fetal distal lung epithelial (FDLE) monolayers. Furthermore, amiloride-sensitive membrane conductance and the open probability of epithelial Na⁺ channels (ENaC) were determined by patch clamp experiments using A549 cells.

Key Results

I_SC was increased by approximately 50% after addition of 1-EBIO, DC-EBIO and chlorzoxazone. With permeabilized basolateral membranes in the presence of a 145:5 apical to basolateral Na⁺ gradient, the benzimidazolones markedly increased amiloride-sensitive I_SC. 5-(N-Ethyl-N-isopropyl)amiloride-induced inhibition of I_SC was not affected. The benzamil-sensitive I_SC was increased in benzimidazolone-stimulated monolayers. Pretreating the apical membrane with amiloride, which inhibits ENaC, completely prevented the stimulating effects of benzimidazolones on I_SC. Furthermore, 1-EBIO (1 mM) and DC-EBIO (0.1 mM) significantly increased (threefold) the open probability of ENaC without influencing current amplitude. Whole cell measurements showed that DC-EBIO (0.1 mM) induced an amiloride-sensitive increase in membrane conductance.

Conclusion and Implications

Benzimidazolones have a stimulating effect on vectorial Na⁺ transport. The antagonist sensitivity of this effect suggests the benzimidazolones elicit this action by activating the highly selective ENaC currents. Thus, the results demonstrate a possible new strategy for directly enhancing epithelial Na⁺ transport.     

Lithium transport by mouse neuroblastoma cells

Lithium entry and its distribution into veratridine-activated cells of an electrically excitable clone (N1E-115) of mouse neuroblastoma differed from that observed in resting cells. At a constant external lithium concentration, increasing the concentration of veratridine (an alkaloid which selectively activates sodium channels responsible for the generation of action potentials), resulted in a corresponding increase of both the rate of entry as well as the intracellular steady state concentration of lithium. Lithium entry occurred by a temperature dependent (Q₁₀ ～- 1.5), formally saturable process having an apparentK_T of about 20 mM and an apparent V_max which increased from 3 nmol · (mg protein) ^?1 · (min)^?1 in the absence of veratridine to 14 nmol · (mg protein)^?1 · (min)^?1 in the presence of 0.67 mM veratridine. In both the veratridine-activated and resting states, mouse neuroblastoma cells concentrated lithium from the external medium, achieving steady state levels within 60 min; thus, in the absence of veratridine. neuroblastoma cells exhibited an intra: extracellular lithium distribution ratio of about 1.5 compared to a distribution ratio of 3.3 in the presence of 0.1 mM veratridine. Lithium accumulation in the presence of supranormal concentrations of extracellular potassium was not different from that found in controls. In addition to characterizing the differences between lithium transport found in veratridine-activated and resting cells, these results suggest that electrically active tissue may accumulate lithium to an extent proportional to the activity of the tissue.     

Amino acid-conferred protection against melphalan—Characterization of melphalan transport and correlation of uptake with cytotoxicity in cultured L1210 murine leukemia cells

The uptake of melphalan by L1210 cells was reduced to one-sixth of controls by physiological concentrations of the l-isomers of leucine and glutamine, and this decrease was accompanied by a corresponding decrease in cytotoxicity. Cytotoxicity was estimated by treatment of cells with melphalan for 35 min in phosphate-buffered saline containing bovine serum albumin and glucose followed by clonal growth of the surviving cells in soft agar. It was prominent within a critical region of net melphalan uptake of 2–5 pmoles/10⁵ cells. Inhibition analysis revealed that at cytotoxic concentrations melphalan is transported by a high-affinity amino acid transport system of the leucine (l) type. The values of the Michaelis constants (K_m) for l-leucine, a protective amino acid, l-valine, a minimally protective amino acid, and melphalan were 6, 58 and 19 μM respectively. These results suggest that the ability of amino acids to protect L1210 cells from melphalan cytotoxicity is related to their affinities for the leucine carrier sites.     

Perchlorate transport and inhibition of the sodium iodide symporter measured with the yellow fluorescent protein variant YFP-H148Q/I152L

Perchlorate is an environmental contaminant that impairs thyroid function by interacting with the sodium iodide symporter (NIS), the transporter responsible for iodide uptake in the thyroid gland. Perchlorate is well known as a competitive inhibitor of iodide transport by NIS, and recent evidence demonstrates that NIS can also transport perchlorate. In this study, we evaluated the yellow fluorescent protein (YFP) variant YFP-H148Q/I152L, as a genetically encodable biosensor of intracellular perchlorate concentration monitored by real-time fluorescence microscopy. Fluorescence of recombinant YFP-H148Q/I152L was suppressed by perchlorate and iodide with similar affinities of 1.2 mM and 1.6 mM, respectively. Perchlorate suppressed YFP-H148Q/I152L fluorescence in FRTL-5 thyroid cells and NIS-expressing COS-7 cells, but had no effect on COS-7 cells lacking NIS. Fluorescence changes in FRTL-5 cells were Na⁺-dependent, consistent with the Na⁺-dependence of NIS activity. Perchlorate uptake in FRTL-5 cells resulted in 10-fold lower intracellular concentrations than iodide uptake, and was characterized by a higher affinity (K_m 4.6 μM for perchlorate and 34.8 μM for iodide) and lower maximal velocity (V_max 6.8 μM/s for perchlorate and 39.5 μM/s for iodide). Perchlorate also prevented iodide-induced changes in YFP-H148Q/I152L fluorescence in FRTL-5 cells, with half-maximal inhibition occurring at 1.1-1.6 μM. In conclusion, YFP-H148Q/I152L detects perchlorate accumulation by thyroid and other NIS-expressing cells, and reveals differences in the kinetics of perchlorate versus iodide transport by NIS.     

Luminal transport of thiol S-conjugates of methylmercury in isolated perfused rabbit renal proximal tubules

Lumen-to-cell transport, cellular accumulation, and toxicity of l-cysteine (Cys), glutathione (GSH) and N-acetylcysteine (NAC) S-conjugates of methylmercury (CH₃Hg⁺) were evaluated in isolated, perfused rabbit proximal tubular segments. When these conjugates were perfused individually through the lumen of S₂ segments of the proximal tubule it was found that Cys-S-CH₃Hg and GSH-S-CH₃Hg were transported avidly, while NAC-S-CH₃Hg was transported minimally. In addition, 95% of the ²⁰³Hg taken up by the tubular cells was associated with precipitable proteins of the tubule, while very little was found in the acid-soluble cytosol. No visual cellular pathological changes were observed during 30 min of study. Luminal uptake of Cys-S-CH₃Hg was temperature-dependent and inhibited significantly by the amino acids l-methionine and l-cystine. Rates of luminal uptake of GSH-S-CH₃Hg were twice as great as that of Cys-S-CH₃Hg and uptake was inhibited significantly (74%) by the presence of acivicin. When 2,3-bis(sulfanyl)propane-1-sulfonate (DMPS) was added to the bathing or luminal fluid, luminal uptake of Cys-S-CH₃Hg was diminished significantly. Overall, our data indicate that Cys-S-CH₃Hg is likely a transportable substrate of one or more amino acid transporters (such as system B^0,+ and system b^0,+) involved in luminal absorption of l-methionine and l-cystine along the renal proximal tubule. In addition, GSH-S-CH₃Hg appears to be degraded enzymatically to Cys-S-CH₃Hg, which can then be taken up at the luminal membrane. By contrast NAC-S-CH₃Hg and Cys-S-CH₃Hg (in the presence of DMPS) are not taken up avidly at the luminal membrane of proximal tubular cells, thus promoting the excretion of CH₃Hg⁺ into the urine.     

Blood-to-retina transport of riboflavin via RFVTs at the inner blood-retinal barrier

Riboflavin (vitamin B₂) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [³H]riboflavin (62.8 μL/(min·g retina)) was much higher than that of a non-permeable paracellular marker, suggesting the facilitative influx transport of riboflavin across the BRB. The retinal uptake index (RUI) of [³H]riboflavin was 59.0%, and significantly reduced by flavin adenine dinucleotide (FAD), but not by l-ascorbic acid, suggesting the substrate specificity of riboflavin transport. TR-iBRB2 cells, an in vitro model of the inner BRB, showed a temperature- and concentration-dependent [³H]riboflavin uptake with a K_m of 113 nM, suggesting that the influx transport of riboflavin across the inner BRB involves a carrier-mediated process. [³H]Riboflavin uptake by TR-iBRB2 cells was slightly altered by Na⁺- and Cl⁻-free buffers, suggesting that riboflavin transport at the inner BRB is preferentially Na⁺- and Cl⁻-independent. [³H]Riboflavin uptake by TR-iBRB2 cells was significantly reduced by riboflavin analogues while the uptake remained unchanged by other vitamins. The function and inhibition profile suggested the involvement of riboflavin transporters (SLC52A/RFVT) in riboflavin transport at the inner BRB, and this is supported by expression and knockdown analysis of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3) in TR-iBRB2 cells.     

Tight binding inhibitors--VIII. Studies of the interactions of 2'-deoxycoformycin and transport inhibitors with the erythrocytic nucleoside transport system

Studies were performed to extend earlier observations that the rate-limiting step in the inactivation of intraerythrocytic human adenosine deaminase (ADA) by 2'-deoxycoiormycin (dCF) is the nucleoside transport system (NTS). The NTS inhibitors 2-amino-6-[(2-hydroxy-5-nitrobenzyl)thio]-9-β-D-ribofuranosyl purine (HNBTGR), 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosyl purine (NBMPR), 2-amino-6-[(4-nitrobenzyl)Seleno]-9-β-D-ribofuranosyl purine (NBSeGR), dipyridamole and the competitive permeant, uridine, all decreased the rate of ADA inactivation by dCF in a concentrationdependent manner. Lineweaver-Burk plots of $\text{1}\text{k}{}_{\mathrm{\gamma }}$ (where k_γ is the pseudo first-order rate constant for the inactivation of ADA) 1/dCF concentrations were linear, giving a K_m tor dCF tor the NTS of 6 × 10^?7 M. The maximal k_γ calculated by extrapolation to infinite dCF concentrations was 6 × 10^?3 per sec which corresponds to a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of about 115 sec. Similar plots for experiments with the NTS inhibitors and uridine yielded classic patterns of competitive inhibition for NBMPR, HNBTGR, NBSeGR and uridine, whereas with dipyridamole a pattern of non-competitive inhibition was obtained. Dissociation or inhibition constants have been reported for several of these compounds (determined by other methods) and values similar to these were obtained. Inhibition by dipyridamole was non-competitive (k_l = 2.5 × 10^?7 M) and was of a bi-phasic nature with respect to time. Dipyridamole caused rapid and irreversible inhibition for the first 7–15 min with slow and progressive but reversible inhibition thereafter. These observations are consistent with the hypothesis that NBMPR, HNBTGR, NBSeGR and uridine interact with the same site on a macromolecular component of the NTS that forms ligands with dCF. The behavior of dipyridamole appears more complex and will require more extensive study.     

Interaction of central nervous system drugs with synaptosomal transport processes.

Interaction of a wide range of central nervous system (CNS) drugs with the transport of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) into synaptosomes from cerebral cortex was investigated. By determining the effect on Na,K-ATPase, the interaction of these drugs with the synaptic transport of Na ⁺ and K⁺ was also assessed. Although of different numerical value, the order of potency of the drugs in inhibiting the uptakes of both biogenic amines was comparable. Furthermore, similar inhibition constants were obtained for the low affinity uptake of 5-HT and the uptake of NE. Desipramine and amphetamine strongly inhibited both uptake processes. The narcotic drugs displayed a wide range of inhibitory potency. An exceptionally strong, but not stereospecific, effect was obtained with levorphanol, which inhibited 5-HT uptake with a K_i of 0.7 μM. While methadone and pentazocine had inhibition constants between 20 and 50 μM, morphine, codeine and naloxone were markedly weak inhibitors with K_i, values extending into the mM range. The uptake of both 5-HT and NE was strongly inhibited by quinacrine. Among the drugs investigated, only methadone showed substantial inhibition of synaptosomal Na,K-ATPase. The reversible inhibition of the enzyme by the drug was diminished at concentrations of Na ⁺ and K ⁺ reflecting synaptic discharge. The drug partially bound to the ouabain site of Na,K-ATPase and also inhibited the Mg-activated component. The feasibility and experimental conditions were established for using a modified crude mitochondrial fraction as synaptosomal preparation to study transport processes in general and the uptake of biogenic amines in particular.     

Benzylpenicillin transport and subcellular distribution in mouse peritoneal macrophage monolayers

[¹⁴C]Benzylpenicillin was rapidly taken up into and eluted from mouse peritoneal macrophage monolayers and entered cytosol, lysosomes and phagolysosomes containing Staphylococcus aureus. Equilibrium concentrations in cytosol and lysosomes were consistent with transport by diffusion but partial membrane carrier dependence was evidenced by saturability (K_m and V_max 110 mM and 117 nmoles (10⁶ cells)^?1 (3 min)^?1 respectively) and inhibition by probenecid. Partial dependence of transport on metabolic energy was shown by inhibition with cyanide plus 2-deoxyglucose and the temperature coefficient (Q₁₀) was about 2. Some of the intralysosomal radiolabel accumulated as benzylpenicilloic acid.     

Arsenate transport by sodium/phosphate cotransporter type IIb

Arsenic is a metalloid that causes the dysfunction of critical enzymes, oxidative stress, and malignancies. In recent years several transporters of As^III have been identified, including aquaglyceroporins (AQP) and multidrug resistance proteins (MRP). As^V transport, however, has not been sufficiently studied because it has been assumed that arsenate is taken up by mammalian cells through inorganic phosphate (Pi) transporters. In this paper we have analyzed the role of Pi transporters in the uptake of arsenate by directly using ⁷³As^V as a radiotracer in phosphate transporter-expressing Xenopus laevis oocytes. The affinities of Pi transporters for H₃AsO₄ were lower than the affinities for Pi. NaPiIIa, NaPiIIc, Pit1, and Pit2 showed a K_m for arsenate that was > 1 mM (i.e., at least ten times lower than the affinities for Pi). The NaPiIIb isoform showed the highest affinity for As^V in mouse (57 μM), rat (51 μM), and human (9.7 μM), which are very similar to the affinities for Pi. Therefore, NaPiIIb can have a prominent role in the toxicokinetics of arsenic following oral exposure to freshwater or food contaminated with As^V.     

Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotomus versicolor.

NADPH-cytochrome c reductase, benzo[a]pyrene hydroxylase and aminopyrine demethylase activities in hepatic microsomes from the marine fish scup (Stenotomus versicolor) were characterized according to dependence of Ph, temperature, ionic strength and Mg²⁺. The kinetic properties of benzo[a] pyrene hydroxylase were variable, depending on protein and substrate concentration, with measured K_m values for benzo[a]pyrene between 4 × 10^?7 M and 4 × 10^?5 M. The K_m for aminopyrine was 7 × 10^?4 M, and NADPH-cytochrome c reductase had K_m values of 2.1 × 10^?5 M and 1.3 × 10^?5 M for cytochrome c and NADPH. respectively. NADH supported benzo[a]pyrene hydroxylation at 10 per cent of the rate seen with NADPH, and no synergism was observed. Aminopyrine demethylation proceeded at least as well with NADH as with NADPH, and there was synergism when combined. NADPH- and NADH-cytochrome c reductases were detected in “microsomes” from fourteen extrahepatic tissues, including kidney, testis, foregut, gill, heart, red muscle, hindgut, buccal epidermis, pyloric caecum, spleen, brain, lens, ovary and white muscle. Benzo[a]pyrene hydroxylase was detected in all but white muscle, while cytochrome P-450 and aminopyrine demethylase were detectable in fewer tissues. Reduced, CO-ligated absorption maxima in the Soret region were 450 nm for all those but liver (occasionally 449 nm) and heart (about 447 nm). The estimated turnover numbers for benzo[a]pyrene hydroxylase and aminopyrine demethylase, and the influence of 7,8-benzoflavone in vitro on benzo[a]pyrene hydroxylase indicate that the cytochromes P-450 in different fish tissues are not catalytically equivalent.     

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na(+) transport across human airway epithelial cells

BACKGROUND AND PURPOSE

Pulmonary transepithelial Na⁺ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na⁺ channels and basolateral Na⁺K⁺ ATPase activity.

EXPERIMENTAL APPROACH

H441 human airway epithelial cells were used to examine the effects of hypoxia on Na⁺ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS.

KEY RESULTS

AMPK was activated by exposure to 3% or 0.2% O₂ for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm⁻²) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O₂ activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I_sc (I_ouabain) and apical amiloride-sensitive Na⁺ conductance (G_Na+). Modification of AMPK activity prevented the effect of hypoxia on I_ouabain (Na⁺K⁺ ATPase) but not apical G_Na+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G_Na+ (epithelial Na⁺ channels).

CONCLUSIONS AND IMPLICATIONS

Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na⁺ channels and basolateral Na⁺K⁺ ATPase activity to decrease transepithelial Na⁺ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.     

Drug delivery studies in Caco-2 monolayers. III. Intestinal transport of various vasopressin analogues in the presence of lysophosphatidylcholine

The transport of a series of vasopressin and oxytocin analogues with varying lipophilicities was studied in Caco-2 monolayers. Transport was studied across the bare monolayer and after treatment with a phospholipid absorption enhancer, palmitoyl lysophosphatidylcholine. The range in lipophilicity of the analogues, estimated as the capacity factor, was found to be from 0.19 to 3.43. The intrinsic transport of the peptides across Caco-2 monolayers was found to be low. The apparent permeability coefficients, P_app, were in the range of 2 × 10⁻⁸–6 × 10⁷ cm/s. However, peptide transport was significantly greater (P_app in the range of 5 × 10⁻⁶–2 × 10⁻⁵ cm/s) when facilitated by addition of palmitoyllysophosphatidylcholine. The results suggest that polypeptide transport across Caco-2 monolayers does not depend on lipophilicity, but that the facilitated transport does depend on the lipophilicity.     

Comparison of the properties of phosphoribosylpyrophosphate synthetase in normal and leukemic human white blood cells.

Phosphoribosylpyrophosphate synthetase (PRPP synthetase) from normal human lymphocytes and granulocytes was compared with phytohemagglutinin (PHA)-stimulated lymphocytes and leukemic white blood cells of several types with respect to enzyme activity and kinetic constants. PRPP synthetase activity was determined by measuring the production of ¹⁴CO₂ in a coupled reaction with ¦¹⁴C¦orotic acid in the presence of orotidylate pyrophosphorylase and orotidylate decarboxylase. Enzyme activity was expressed both as nmoles/mg of protein/30 min and as nmoles/10³ cells/30 min. In more than 50 per cent of the leukemic patients, activity was above normal values when compared on a per mg protein basis. However, when activity was compared on a per cell basis, only the acute myelocytic leukemic patients showed a change from the normal value, and in this case activity was inversely related to the percentage of blast cells in the peripheral circulation. Michaelis constants for ATP (K_m^ATP) and ribose-5-phosphate (K_m^R5P) were found to be 17.6 ± 1.8 and 51.3 ± 2.4 μM, respectively, in normal lymphocytes, and 55.9 ± 8.0 and 82.5 ± 1.1 μM in normal granulocytes. The K_m^ATP was found to decrease in all leukemic cell types, while the K_m^R5P showed deviation from normal values depending on the type of leukemia. The inhibition constants (K_i^ATP and K_i^R5P) were also compared in all leukemic cell types and showed deviations from normal which were cell type dependent. These results suggest that sufficient alteration of the properties of PRPP synthetase from leukemic cells exists to warrant further investigation into the therapeutic implications of alterations of the properties of this enzyme.