Metabolic Energy Supply Research Articles

Uptake of the food-derived heterocyclic amine carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and its N-hydroxy metabolite into rat pancreatic acini and hepatocytes in vitro.

Sinc DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are formed at relatively high levels in the rat pancreas but not liver, we examined the uptake of PhIP and its N-hydroxy metabolite (N-OH-PhIP) into pancreatic acini and hepatocytes to determine if differential tissue uptake was a factor modulating the formation of PhIP-DNA adducts. In addition, since the precursors of PhIP formation are two amino acids and since various amino acid transporters have been identified in the pancreas, the possible involvement of these transporters in the uptake of PhIP and N-OH-PhIP was investigated. The uptake both heterocyclic compounds into both tissue preparations was rapid, with maximal uptake occurring with 1-2 min. However, PhIP uptake into pancreatic acini was significantly (2-way ANOVA, P < 0.05) greater than uptake of N-OH-PhIP into pancreatic acini and the uptake of both PhIP and N-OH-PhIP into hepatocytes. Although uptake was rapid, efflux of both compounds from both tissue preparations was also rapid. However, the efflux rate constant (1.86 +/- 0.6/min, mean +/- SEM) for PhIP) was significantly lower (Student's t-test, P < 0.05) than that for N-OH-PhIP (4.14 +/- 0.04/min) from pancreatic acini. This, combined with the increased uptake of PhIP into pancreatic acini , suggests that there is substantial but reversible binding of PhIP in the pancreas. The uptake of both PhIP and N-OH-PhIP into pancreatic acini and hepatocytes was not affected by the presence of various amino acids in the incubation buffer, indicating that amino acid transporters are not involved in uptake of these compounds. Furthermore, uptake of both compounds did not appear to be dependent on metabolic energy supply. The above data, together with the high octanol:buffer partition coefficients (logP = 1.322 and 1.301 for PhiP and N-OH-PhIP respectively) suggest that both uptake and efflux of PhIP and N-OH-PhIP are consistent with a process of passive diffusion. The tissue binding characteristics for PhIP in the pancreas may create conditions whereby pancreatic cytochrome P450 1A1 can catalyse the formation of N-OH-PhIP. While N-OH-PhIP is not the ultimate reactive DNA binding species, it has been shown to directly bind to and form DNA adducts. Therefore, it is possible that the apparent selective accumulation of PhIP may contribute to the high level of PhIP-DNA adducts formed in the rat pancreas.

Dependence of receptor potential and redox state of mitochondrial cytochromes on oxygen fraction measured in the blowfly eye in vivo

1. The dependence of dark-adapted fly (Calliphora vicina) photoreceptors on oxygen was investigated by measuring the electroretinogram (ERG), the receptor potential, and the redox states of the mitochondrial cytochromes. The redox states were determined via reflection microspectrophotometry on white-eyed fly mutants. 2. The light sensitivity of the photoreceptors at oxygen fractions above ≈ 2% is identical to that in the normal (air) environment, as judged from both ERG and receptor potential. Light sensitivity is rather abruptly and strongly reduced at oxygen fractions lower than ≈ 1% and vanishes at anoxia (0%). 3. The redox state of the mitochondrial cytochromes also changes around oxygen fractions of 1–2%, but the dependence on oxygen fraction is less abrupt than that of ERG and receptor potential. 4. The mitochondrial activity of dark-adapted fly photoreceptors appears to be well-buffered, to warrant ample supply of metabolic energy for unimpaired photoreceptor function down to extremely low oxygen levels.

Respiratory Characteristics inPyricularia oryzaeExposed to a Novel Alkoxyiminoacetamide Fungicide

(E)-2-Methoxyimino-N-methyl-2(2-phenoxyphenyl) acetamide (SSF-126) inhibited respiration of Pyricularia oryzae mycelia by blockade of the electron flux through the cytochrome bc 1 segment of the mitochondrial respiratory chain. However, the mycelia again began to respire at 20 min after treatment. The recovered respiration was sensitive to salicylhydroxamic acid, a potent inhibitor of cyanide-resistant respiration, but not to potassium cyanide, indicating that SSF-126 had the ability to induce cyanideresistant respiration in P. oryzae. The recovered respiration was partially linked to energy transduction because SSF-126 could not completely suppress the mycelial growth. SSF-126 caused a rapid depletion of ATP content, but the ATP levels showed recovery, resulting in improvement of energy charge values. However, the total adenylate pool gradually decreased, resulting in only 60% remaining 48 h after treatment. SSF-126 markedly suppressed the mycelial growth, the duration for mass doubling in cultures incubated with SSF-126 being 2.4 times that in the control ones. Thus, SSF-126 works deleteriously on the supply of metabolic energy in P. oryzae

Expression patterns of ammonia-metabolizing enzymes in the liver, mesonephros, and gut of human embryos and their possible implications.

Human and ungulate embryos can catabolize amino acids for energy production, whereas rodent embryos cannot, raising the question whether studies of rodent model systems are suitable for extrapolation to the human situation. Therefore, we investigated the expression of the amino acid- and ammonia-metabolizing enzymes glutaminase, glutamate dehydrogenase, glutamine synthase, carbamoylphosphate synthase, and arginase immunohistochemically in a graded series of human embryos and fetuses. During human development the expression of these enzymes is first seen in the liver, then in the mesonephric kidney, and finally in the small intestine. Such a simultaneous expression of nitrogen-metabolizing enzymes was not seen in any other organ. The early appearance of the enzymes involved in amino acid and ammonia metabolism in the human liver, compared to, for example, the rat liver, suggests that catabolism of amino acids may provide an important supply of metabolic energy for the human embryo. The coexpression of glutaminase, glutamate dehydrogenase, and carbamoylphosphate synthase, but not of arginase, in the mesonephros and the small intestine suggests that these organs are involved in the biosynthesis of intermediates of the ornithine cycle, e.g., arginine or citrulline. From a comparison of the developmental appearance of ornithine cycle enzymes in different mammalian species we postulate that an early appearance of these enzymes is generally associated with a relatively slow prenatal growth rate and the use of amino acids as metabolic fuel.

Lidocaine, a local anesthetic, reversibly inhibits cytoplasmic Streaming inVallisneria mesophyll cells

Lidocaine, which like other local anesthetics is known to inhibit intracellular transport in animal cells, was tested for its effect on the rotational cytoplasmic streaming in the mesophyll cells of the aquatic plantVallisneria. The drug caused reversible inhibition of cytoplasmic streaming in a dose dependent manner within the 2–20 mM range; higher concentrations resulted in permanent cessation of all cytoplasmic motion. Upon recovery following replacement of the normal bathing medium, cytoplasmic rotation was always resumed in the direction of the original movement exhibited by a given cell. The lidocaine effect was virtually independent of the ionic composition of the incubation medium, but it was markedly affected by the external pH; acidic conditions (pH 6) largely prevented the inhibition of streaming, whereas an alkaline environment (pH 8) accelerated both the onset of the effect and the recovery upon removal of the anesthetic. On the basis of these results and findings in other systems, it is suggested that lidocaine acts through interference with mechanisms that regulate cytoplasmic streaming, rather than with the motile apparatus or the supply of metabolic energy.

Effects of ambient temperature and body fat content on maternal litter reduction in Syrian hamsters

Reproduction in Syrian hamsters is sensitive to the general availability of metabolic energy. For example, females often modify their litter size by cannibalism on days 1–7 postpartum, and the number of young eaten is a function of the total supply of metabolic energy as determined by both food supply and body fat content. If the level of cannibalism is a function of energy availability, it might be expected that a drop in ambient temperature would increase cannibalism, since cold acclimation demands greater energy expenditure. We found that hamsters ate significantly more of their offspring when housed at 10 compared to 22°C during lactation. The effect of cold on cannibalism was attenuated in hamsters fattened prior to cold exposure and exaggerated in hamsters that were lean prior to cold exposure. Thus, the litter size maintained by Syrian hamsters is a function of the total supply of metabolic fuels as determined by energy sources, such as food supply and adipose tissue, and by energetic costs of thermoregulatory and other processes.

Spreading of trypsinized cells: Cytoskeletal dynamics and energy requirements

The spreading of trypsinized XTH-2 cells (a line derived from Xenopus laevis tadpole heart endothelia) on glass was investigated. Three phases can be distinguished: (1) blebbing of rounded cells, first attachment to a solid substratum and formation of a broad smooth contact area; (2) organization of a peripheral zone of actin fibrils and reinforcement of the basal cytoplasm by a stress fibre-like pattern; (3) extension of lamellae. The first phase seems to be independent of a supply of metabolic energy, while the others clearly depend on it. This is concluded from the close relationship between cellular projection area and energization of mitochondria as revealed by (a) the fluorescence intensity of cells vitally stained with the mitochondria-specific fluorochrome DASPMI (2-4-(dimethylamino)-styryl-1-methylpyridinium-iodine); (b) the degree of spreading in the presence of inhibitors of respiration; (c) effective amelioration of spreading (phases (2) and (3] under conditions of high ATP content. In phase (2) the extension of the central part of the cells becomes stabilized, the cell body settles on the basal cytoplasmic layer and further expansion of the projection area is achieved by lamella formation (phase (3]; motile and stabile regions of the cells become separated. This sequence of events is interpreted as a self-organizing process based on the development of internal hydraulic pressure, actin polymerization and contraction of the newly developed actomyosin network. During trypsinization, depolymerization of actin does not occur but rather on addition of Ca2(+)-containing media. Cellular ATP content drops as well on trypsinization, as on addition of Ca2+. Manganese promotes spreading by decreasing F-actin disassembly and maintaining a high level of cytosolic ATP, most probably because it is not accepted by the calcium pumps. Regarding the association of glycolytic enzymes with F-actin and their influence on actin assembly, lactate dehydrogenase has been inhibited with oxamic acid. This treatment improves the correlation between F-actin content and the degree of spreading; however, the total amount of F-actin remains smaller and the cells spread more.

Effects of maternal diet, body weight and body composition on infanticide in Syrian hamsters

We examined the idea that female hamsters kill and eat their own offspring as part of an organized mechanism that balances litter size with metabolic energy supply. In Experiment 1, females fed diets that made them lighter and leaner cannibalized more offspring and maintained smaller litters than females fed diets that made them heavier and fatter. A greater supply of metabolic energy from the diet and/or from body fat stores may have attenuated cannibalism in heavier mothers. In Experiment 2, food restriction during lactation increased the level of cannibalism to a greater degree in lighter, leaner mothers. Heavier, fatter mothers may have eaten fewer offspring because they were better able to mobilize fatty acids from adipose tissue as an alternative fuel source during food restriction. These results suggest that an important factor influencing cannibalism of pups is the general availability of metabolic fuels from both external (food supply) and internal (adipose tissue) sources.

Anthracycline-induced inhibition of membrane permeability functions dependent on metabolic energy.

The influence of anthracyclines on membrane permeability functions has been investigated in HeLa cells by monitoring the efflux of fluorescein. Release of the fluorescent dye, dependent on the metabolic energy supply, occurs after the intracellular accumulation and enzymatic hydrolysis of the non-fluorescent substrate fluorescein diacetate (FDA). Flow cytometric evaluation of the efflux kinetics showed that adriamycin (ADR), N-trifluoroacetyladriamycin-14-valerate (AD-32) and daunorubicin (DNR) inhibited the permeability process. The degree of inhibition was dependent, though to different extent, on the intracellular concentration of each drug. An increase in the efflux rate was always observed when the cells were treated with the drugs in the presence of 20 mM glucose. Relationship of these effects with energetic metabolism was supported by the finding that ATP levels were lowered by the drugs and increased by glucose. Evaluation of the cytotoxicity induced by each drug showed that the intracellular amount necessary to inhibit cell survival by 50% was of the same order of magnitude as that which decreases to 50% membrane permeability to fluorescein. These results indicate a correspondence in the concentrations of anthracyclines required for inducing cytotoxicity and for inhibiting membrane permeability functions dependent on the metabolic energy supply.

Facilitated glucose transport across the retinal pigment epithelium of the bullfrog ( Rana catesbiana)

Transport studies of glucose analogs [3H] 3-O-methyl-D-glucose (mD-glu) and L-[14C]glucose (L-glu) across the isolated retinal pigment epithelium (RPE) of the bullfrog was undertaken to determine whether the glucose transport mechanism was dependent upon the postulated ion-transport scheme and/or whether glucose transport is insulin-mediated. In addition, metabolic inhibitors were tested to explore the energy requirements of glucose transport across the RPE. Flux studies of mD-glu and L-glu performed with mounted RPE tissues, with short circuit current (SCC) and potential difference (PD) monitored via automatic voltage clamp apparatus, indicate that transport is clearly stereospecific with D-glucose being transported at least 13 times faster than L-glucose. The system was found to be saturable with a Km of about 24 mM glucose and Vmax of 1400 nmol cm-2 hr-1. Unidirectional Michaelis-Menten constants indicate that the RPE glucose carrier is accessible for transport from either the choroid or retinal side and a bidirectional facilitated diffusion mechanism is suggested. Insulin had no effect on either ion transport (SCC) or glucose transport (passive or facilitated). Both aerobic and anaerobic energy inhibitors decreased ion transport to less than 25% of control, but had little effect, if any, on glucose transport across the isolated RPE. Sodium iodoacetate decreased ion transport by 90% of control, but a much slower decrease in facilitated glucose transport of 22% of control suggests that carrier energy requirements, if any, are not direct or immediate. Osmotic studies performed with sucrose and glucose suggest that elevations in osmolarity increase passive glucose movement and decrease facilitated glucose-transport rates. Glucose was found to be much more detrimental to glucose transport than sucrose, suggesting that at high concentrations molecular glucose decreases facilitated transport and increases passive glucose movement by a mechanism other than can be accounted for by osmotic considerations. A model for RPE glucose transport, consistent with current data, is proposed which translocates D-glucose, via an alternating conformational change of the glucose carrier. This carrier does not require a direct supply of metabolic energy, nor a functioning ion-transport mechanism. At a given moment, a single binding site for D-glucose is postulated to be available on either side of the RPE membrane for glucose translocation, although binding site affinity for glucose could differ on each side.

Balanced contribution of glycolytic and adenylate pool in supply of metabolic energy in platelets.

When platelets are treated with H2O2 the metabolic ATP content decreases sharply (Holmsen, H., and Robkin, L. (1977) J. Biol. Chem. 252, 1752-1757). Here we report that the loss of metabolic energy is fully recovered in phosphorylated glycolytic intermediates. A mixture of antimycin A/2-deoxy-D-glucose/D-gluconic acid-1,5-lactone blocks mitochondrial ATP resynthesis and prevents the entry of sugars into the glycolytic sequence. The energy-rich phosphates in the adenylate and the glycolytic pool are then consumed in a specific order. First, the glycolytic pool is consumed at a rate of 4.5 mumol of ATP equivalents/min/10(11) cells, and metabolic ATP and ADP are kept stable; then the consumption of the glycolytic pool decreases and metabolic ATP and ADP are consumed, together keeping up with the same rate of energy consumption. Thrombin stimulation increases the energy consumption to about 17 mumol of ATPeq/min/10(11) cells which is now furnished by both the glycolytic and the adenylate pool, again with a preferential consumption of the former. The results show that H2O2 triggers a shift of energy-rich phosphates from the adenylate to the glycolytic pool and that the latter remains rapidly accessible to energy consumption thereby stabilizing the level of metabolic ATP. The adenylate energy charge is independent of the distribution of energy among the two pools, which extends its importance to the regulation of energy supply and demand beyond the adenylate pool.

Energy consumption in a cyclic phosphorylation/dephosphorylation cascade.

Cyclic phosphorylation/dephosphorylation cascade systems are responsible for regulating numerous metabolic pathways. The capacity of a cyclic cascade system to maintain a steady-state level of phosphorylation and, hence, a specific biological activity of a phosphorylatable protein is dependent upon a constant supply of metabolic energy (ATP). Quantification of the extent of ATP consumption in a cyclic cascade was examined experimentally with the model in vitro phosphorylation/dephosphorylation system described in detail in the previous paper (Shacter, E., Chock, P. B., and Stadtman, E. R. (1984) J. Biol. Chem. 259, 12252-12259). The results indicate that (a) when the concentrations of converter enzymes and interconvertible substrate are held constant and the fractional phosphorylation of the substrate is varied by changing the allosteric effector concentrations, the rate of ATP consumption in the monocyclic cascade is directly proportional to the steady-state level of phosphorylation being maintained. (b) Attainment of a particular steady-state level of phosphorylation is determined by the net ratio of the protein kinase and phosphatase activities and is independent of the absolute concentrations of these enzymes. (c) Whereas the time required to reach a given steady state is inversely proportional to the converter enzyme concentrations, the amount of ATP consumed in maintaining that steady state is directly proportional to the kinase and phosphatase concentrations. In addition, a theoretical analysis based upon experimentally determined parameters for two in vivo cyclic cascade systems (pyruvate kinase and glycogen phosphorylase) revealed that under normal conditions, cyclic phosphorylation/dephosphorylation cascades consume only a small proportion (less than 0.02%) of the total cellular energy flux.

Characterization of valine transport in sea urchin eggs

In unfertilized eggs, the mechanism of valine uptake can be summarized as follows. It is saturable over the external concentration of valine and insensitive to the presence of external sodium, depletion of cellular energy supplies and intracellular acidosis. The activation energy for the transport reaction (16.3 kcal/mol) is within the range of values reported for active transport of small molecules. In fertilized eggs, the total rate of valine uptake can be divided into two components: (i) a Na +-insensitive uptake which accounts for about 7% of total absorption as shown by studies in Na +-free medium seems to possess the same characteristics as in unfertilized eggs, (ii) a Na +-dependent transport of valine which constitutes the main entry is formed about 5 min after fertilization. It follows Michaelis-Menten kinetics characterized by 15-fold increase in V max with no change in K m . These two mechanisms have characteristics in common, such as their insensitivity to metabolic energy supply, their energy of activation and their ability to concentrate valine. The relationship between the establishment of the Na +-dependent valine uptake and the ionic events triggered by fertilization is discussed.

Glutamine uptake by isolated rat brain mitochondria

The existence of a specific mechanism for glutamine transport is demonstrated by observation that brain mitochondria swell when incubated in iso-osmolar solutions of this neutral amino acid. There is no proton or ionophore requirement, but a metabolic energy supply increases the rate of swelling. Carrier mediation is strongly suggested by the observation that l-glutamine is transported more rapidly than its d-isomer. Transport is inhibited by ionic mercurials, but not by N-ethylmaleimide, which rules out any link between glutamine uptake and glutamate efflux, α-ketoglutarate and glutamate are powerful inhibitors of glutamine transport. Interactions between the specific glutamine transport mechanism and the intramitochondrial phosphate-dependent glutaminase activity are discussed.

A quantum mechanical interaction of human erythrocytes.

Theory predicts that the membrane potential will polarize membrane molecules and cause them to vibrate coherently at a frequency of approximately 10(11) HZ. If the supply of metabolic energy exceeds a minimum value, membrane phonons may condense their momentum into a single "giant" vibrational mode. At 10(11) HZ ionic screening is small up to distances of approximately a micrometre, so forces of a range several orders of magnitude longer than chemical forces can arise. These forces may be attractive or repulsive depending on frequency. They should occur in every metabolically active membrane and may control macromolecular transport and enzyme-substrate interactions. We find that normal human erythrocytes in plasma form rouleaux faster than Brownian motion predicts. When cells are fixed in glutaraldehyde or are metabolically depleted, or if the membrane potential is brought to zero, the rate of aggregation agrees with Brownian theory. When the metabolically depleted cells are revived or if the membrane potential is restored, then the interaction returns.

ATP stimulation of the sn-glycerol-3-phosphate acyltransferase of Escherichia coli

ATP was found to stimulate the rate of the inner membrane sn-glycerol-3-phosphate acyltransferase of Escherichia coli. Stimulation required the presence of Mg 2+ and was demonstrated with either coenzyme A or acyl carrier protein thioesters as the acyl donor. The ATP stimulation was consistently observed in freshly prepared membranes and those stored at 4 °C, but after freeze/thaw treatment, the acyltransferase no longer responded to ATP. ATP increased the maximal velocity of the reaction but did not affect the Michaelis constants of the substrates. ATP did not drastically alter the proportions or types of products formed in the reaction. The ATP effect may be a mechanism functioning to enhance the rate of the acyltransferase reaction in response to an increased supply of metabolic energy.

The active transport of monosaccharides by the yeast Metschnikowia reukaufii: Evidence for an electrochemical gradient of H + across the cell membrane

The facultative anaerobic yeast Metschnikowia reukaufii was found to take up the monosaccharides d-glucose, d-xylose, and 3- O-methyl- d-glucose by a constitutive mobile membrane carrier. The uptake was dependent on the supply of metabolic energy. The half-saturation constants of transport, K T , amounted to 0.9, 2.0, and 1.0 m m, respectively. Cell suspensions of M. reukaufii catabolized d-glucose and d-xylose, but not 3- O-methyl- d-glucose. d-Xylose was accumulated about 100-fold above the diffusion equilibrium in spite of its catabolism. 3- O-Methyl- d-glucose was accumulated about 40-fold. There was no accumulation of monosaccharides in the presence of uncouplers like carbonylcyanide m-chlorophenylhydrazone or dinitrophenol. For d-glucose utilization, its transport was the ratelimiting step. All three monosaccharides were transported by the same carrier; in a mixture they competed for the binding site at the carrier. An electrochemical gradient of H + ions was measured across the plasma membrane of M. reukaufii cells. The pH difference between the cell interior and the medium of an unbuffered suspension (pH 4–5) reached the value of 2.0 pH units (inside alkaline). The membrane potential measured with the lipid-soluble cation tetraphenylphosphonium at pH 9.0 was about - 100 mV (inside negative). The onset of sugar transport brought about a short alkalinization of unbuffered cell suspensions or a depolarization of the membrane potential in buffered suspensions (pH 9.0). The H +/sugar stoichiometry was one H + ion per each sugar molecule taken up. The data are interpreted as evidence in favor of the active monosaccharide transport in M. reukaufii being an H + symport, energized by the electrochemical gradient of H + across the plasma membrane.

Respiration-dependent Movements of the Column of Stylidium gvaminifolium

The column of the trigger plant, Stylidium graminifolium, when fully set responds to mechanical stimuli by flipping through an angle of about 4 radians in a fast firing movement lasting about 15-30 ms, and then slowly resetting to its original position in about 400 s. After resetting there is an absolute refractory period of about 500 s during which no further response to stimuli can be initiated, followed by a relative refractory period when graded responses increasing in rate and magnitude with time can be obtained. The resetting movement and the process, occurring during the refractory period, allowing subsequent firing to occur, are inhibited when the air surrounding the column is replaced by nitrogen. The firing movement, however, is not affected by these anaerobic conditions. Thus the firing movement is caused by passive physical forces, rapidly utilizing potential energy from a store built up during the previous resetting and refractory periods. Removal of the labellum, which the column touches when set, causes the column to oscillate with amplitude of about 3-3.5 radians and period of 1-2 ks. When the column is held at a constant angular displacement it develops an oscillatory torque with similar period. These oscillations are inhibited at all stages of the cycle by anaerobic conditions. It appears that the oscillatory behaviour is not a slowed-down firing process followed by normal resetting, but is linked throughout the cycle to the metabolic energy supply.

Effects of metabolic intermediates on sugar and amino acid uptake in rabbit renal tubules and brush border membranes.

1. The effects of tricarboxylic acid cycle intermediates on the renal transport of alpha-methyl-D-glucoside and alpha-amino-isobutyric acid were examined using separated renal tubules of the rabbit. 2. The effect of citrate on alpha-methyl-D-glucoside and alpha-amino-isobutyric acid uptake was markedly biphasic with maximum stimulation of transport occurring at a citrate concentration of 0.64 mM. Biphasic effects were also apparent for L-malate, succinate, fumarate, alpha-ketoglutarate and oxaloacetate. 3. The route of uptake of alpha-methyl-D-glucoside into separated renal tubules is primarily across the brush border (luminal) membrane. 4. Tricarboxylic acid cycle intermediates produced significant stimulation of renal O2 consumption; however, the effects on O2 consumption were not biphasic suggesting that reduced stimulation of transport at high substrate concentration was not caused by a reduction in the supply of metabolic energy. 5. In purified renal cortical brush border membrane vesicles, citrate and alpha-ketoglutarate inhibited the uptake of alpha-methyl-D-glucoside and alpha-amino-isobutyric acid indicating that inhibition of their transport in respiring renal tubules by high concentrations of tricarboxylic acid cycle intermediates occurs via an effect at the membrane level.

Active secretion of hypoxanthine and xanthine by guinea pig jejunum in vitro.

Isolated epithelium of guinea pig jejunum secretes hypoxanthine and xanthine by a transport process that is capable of uphill transport and dependent on metabolic energy supply. Unidirectional influx of hypoxanthine across both the luminal and the contraluminal cell membrane appears to be saturable; influx across the contraluminal membrane is inhibited by 2,4-dinitrophenol (DNP). Efflux across the luminal membrane is diminished by DNP; efflux across the contraluminal membrane is increased by DNP. This evidence suggests the existence of a mediated transport system both in the luminal and the contraluminal cell membrane. Additionally, intracellular metabolism of hypoxanthine seems to regulate transepithelial permeation: increased hypoxanthine salvage by the phosphoribosyltransferase reduces the rate of secretion. However, the incorporation of hypoxanthine into the nucleotides is limited when the hypoxanthine is added to the luminal side of the epithelium, and the permeation rate in the absorptive direction is not markedly influenced by the rate of hypoxanthine salvage. These findings are a further example of the functional orientation of the jejunal epithelial cells with respect to enzymic activity and transepithelial transport properties.