Omission Of Glucose Research Articles

Electrical properties of Madin-Darby-canine-kidney cells. Effects of extracellular sodium and calcium.

In incompletely confluent Madin Darby canine kidney cells continuous measurements of the potential difference across the cell membrane (PD) were made with conventional microelectrodes during rapid changes of extracellular sodium and/or calcium concentration. During control conditions PD averages -50.6 +/- 0.7 mV. Reduction of extracellular sodium concentration from 131.8 to 17.8 mmol/l leads to a reversible hyperpolarization of the cell membrane to -65.3 +/- 1.1 mV. This hyperpolarization is not significantly reduced by omission of glucose or presence of amiloride (1 mmol/l) in the perfusates. Instead, 1 mmol/l amiloride depolarizes the cell membrane by +5.2 +/- 0.4 mV. 1 mmol/l barium depolarizes the cell membrane to -31.3 +/- 1.1 mV. Step increases of extracellular potassium concentration from 5.4 to 10 and 20 mmol/l depolarize the cell membrane by +5.5 +/- 0.5 mV and +16.5 +/- 1.8 mV respectively. In the presence of barium, the depolarizing effect of increasing extracellular potassium concentration and of amiloride is almost abolished. Reduction of extracellular sodium concentration in the presence of barium, however, leads to a transient hyperpolarization of the cell membrane. During this transient hyperpolarization, increasing extracellular potassium concentration depolarizes the cell membrane despite the continued presence of barium. Omission of extracellular calcium (EDTA) depolarizes the cell membrane by +36.7 +/- 3.2 mV. In the absence of extracellular calcium, the hyperpolarizing effect of reduced extracellular sodium concentration is markedly reduced (-4.5 +/- 1.2 mV). 2 mumol/l A23187 in the presence of extracellular calcium hyperpolarizes the cell membrane to -72.5 +/- 0.6 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that a "memory' for glucose metabolism desensitizes A-cell responsiveness in the perfused pancreas of the rat.

The effects of prior exposure to glucose or an inhibitor of glycolysis (iodoacetate) on A-cell sensitivity to glucose in the perfused pancreas of the rat was investigated. Inhibition of glucagon secretion by a high glucose concentration (22 mM) was attenuated and delayed when tested 20 min after a previous infusion with the same glucose concentration. Previously elevated glucose also delayed for 2 min a glucagon response to glucose omission whereas the total response was not significantly affected. During a 20 min perfusion with 1 mM iodoacetate, glucagon secretion increased and rates of secretion were further augmented after withdrawal of iodoacetate. When introduced 10 min after cessation of the iodoacetate pulse, 22 mM glucose failed to affect insulin or somatostatin release but, conversely, induced a profound decrease in glucagon secretion which was more marked than during control conditions. A-cell sensitivity to glucose is diminished and enhanced by prior fuel abundance and deprivation, respectively. Such effects could be due to persisting changes in A-cell energy availability rather than to pertubations in insulin or somatostatin secretion.

Luminal and peritubular ionic substitutions and intracellular potential of the rabbit proximal convoluted tubule.

Transepithelial (psi T) and basolateral (psi BL) potential difference was measured in rabbit proximal convoluted tubules perfused in vitro. In control solution without protein, the mean psi BL was -54 +/- 2.2 mV (n = 57). Luminal substitution of K by Na had no effect. Complete luminal substitution of glucose and alanine, 110 mM substitution of Na or NaCl produced transient hyperpolarizations of psi BL of 14, 10, and 13 mV, respectively, with a return close to the control value within 4-8 min in all cases. Returning to control solution produced similar time-course transient depolarizations of psi BL of 17, 11, and 16 mV, respectively, again with a return to the control value in 4-10 min. Omission of glucose and alanine in the perfusate produced a decrease in cell volume of 14% that was maximal in 4 min with a complete recovery in the post-control period. A 110 mM luminal or peritubular substitution of Cl by cyclamate produced no significant effect on psi BL after taking into account the large psi T generated by the diffusion of Cl across the paracellular pathway. On the other hand, complete peritubular substitution of K by Na and 110 mM substitution of Na or NaCl produced sustained but reversible depolarizations of psi BL of 37.5, 10.2, and 20.4 mV, respectively. The transient nature of the hyperpolarization following luminal substitution of glucose, alanine, or Na can be interpreted in terms of changes in the intracellular sodium activity that would affect the Na-K-ATPase pump. Similarly, the sustained depolarization seen after a peritubular substitution of K and Na would also be compatible with a decrease in the basolateral ionic pump activity.

Characteristics of tryptophan accumulation by isolated rat forebrain synaptosomes.

Uptake of 10 microM L-tryptophan into isolated rat brain synaptosomes was studied to assess its effect on the rate of serotonin synthesis from tryptophan. The initial rate of uptake was rapid, being two orders of magnitude above the rate of tryptophan hydroxylation. Uptake was highly concentrative, the concentration ratio across the plasma membrane at equilibrium being approximately 9. This concentration ratio was decreased to about 1 in the presence of high concentrations of amino acids transported by the L-type neutral amino acid uptake system. A mixture of the large neutral amino acids at physiological concentrations decreased the internal tryptophan concentration to 58% of that in their absence. Large tryptophan concentration ratios were observed in experiments in which Na+ in the medium was replaced with choline+. The concentrative uptake of tryptophan was energy-dependent, being decreased by inclusion of cyanide and omission of glucose. The concentration gradient was abolished by veratridine or rotenone. Time courses of the changes in ATP content and tryptophan concentration ratio on addition of these and other agents established that tryptophan uptake is probably not driven by ATP hydrolysis or efflux of other amino acids, but by the plasma membrane potential.

The origin of chemiluminescence produced by neutrophils stimulated by opsonized zymosan.

The luminol-dependent chemiluminescence (CL) of neutrophils phagocytosing zymosan is inhibited by superoxide dismutase (SOD), catalase, sodium benzoate, and 2,5-dimethyl furan. In the present report it is shown that inhibition by SOD and 2,5-dimethyl furan is diminished and removed, respectively, by the omission of glucose from the incubation medium. Zymosan-induced CL is also inhibited by inhibitors of arachidonic acid (AA) metabolism, including 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, quinacrine, indomethacin, and aspirin, by prostaglandins E1 and E2, theophylline, and dibutyryl cyclic AMP (cAMP), and by the addition of AA, sodium fluoride, and xanthine oxidase plus xanthine to the cell suspension. These findings lead us to postulate that the metabolism of AA via the lipoxygenase (and cyclooxygenase) pathway(s) is the source of CL observed in neutrophils after phagocytosis. Reactive oxygen species produced as a result of activation of NAD(P)H oxidase provide oxidizing agents for the oxidation of AA along these pathways. It is also suggested that elevated levels of cAMP induced by prostaglandins synthesized via the cyclooxygenase pathway may play a role in the regulation of the zymosan-induced CL response.

Measurement of sodium ion concentration in the unstirred layer of rat small intestine by polymer Na +-sensitive electrodes

The concentration of sodium ion at the surface of rat small intestine both in vitro and in vivo was measured with plastic polymer sodium ion sensitive electrodes. In vitro the surface sodium ion concentration [Na s +] was found to be significantly higher than bulk bathing solutions of 25 mM concentration. This value could be increased by the addition of glucose to the medium and was significantly higher in the jejunum than in the ileum. Ouabain, deoxycholic acid and dithiothreitol all reduced the [Na s +] under in vitro conditions. In vivo, very high values for [Na s +] were found in the jejunum (approx. 80 mM) when the bulk concentration was 25 mM, indicating a substantial local accumulation of sodium ion at or near the brush border. This could be reduced by omission of glucose from the buffer and further reduced when magnesium was the substituent cation rather than choline. Consequently, in vivo, an appropriately orientated sodium ion gradient persists in the face of adverse tissue to bulk solution concentration gradients and potentially explains why solute absorption occurs under these circumstances. The further reduction of the correctly aligned sodium ion gradient, as required by the gradient hypothesis, by magnesium-substituted buffers indicates that there is no real need to postulate additionally a sodium-independent magnesium-sensitive glucose transport system in vivo.

The effect of cAMP on the cell membrane potential and intracellular ion activities in proximal tubule of Rana esculenta.

Experiments were performed in proximal tubule of the isolated perfused frog kidney to evaluate peritubular cell membrane potentials (PDpt), and the intracellular ion activities of sodium (Nai+), chloride (Cli-) and potassium (Ki+) under control conditions and following peritubular application of dibutyryl-cyclic AMP (cAMP, 2 X 10(-4) mol X 1(-1)). Conventional and ion-sensitive microelectrodes were applied to record continuously cAMP-induced changes of these parameters in individual proximal tubule cells. Within a few minutes a significant hyperpolarisation of PDpt (delta = 2.0 +/- 0.2 mV) occurs simultaneously with a decrease of Nai+ (delta = 2.5 +/- 0.5 mmol X 1(-1)). Ki+ increases (delta = 3.6 +/- 0.9 mmol X 1(-1)) and Cli- decreases (0.4 +/- 0.07 mmol X 1(-1)) slightly, but significantly. With both ions the alterations of the chemical gradient is significantly smaller than the potential shift. PDte is not significantly altered by cAMP. The cAMP-induced hyperpolarisation of PDpt can be observed in presence and absence of luminal glucose. However, omission of Na+ from the luminal perfusate abolishes the hyperpolarising effect of cAMP on PDpt. The results suggest that cAMP reduces sodium entry from the lumen into the cell, thus hyperpolarising the cell membrane and decreasing Nai+. Persistence of sensitivity of PDpt to cAMP after omission of glucose indicates that other Na+ coupled transport processes and/or passive Na+ conductance are affected by cAMP. the changes of Ki+ and Cli- are secondary, following the change of PDpt.

Epinephrine regulation of skeletal muscle glycogen metabolism. Studies utilizing the perfused rat hindlimb preparation.

Studies of rat skeletal glycogen metabolism carried out in a perfused hindlimb system indicated that epinephrine activates phosphorylase via the cascade of phosphorylation reactions classically linked to the beta-adrenergic receptor/adenylate cyclase system. The beta blocker propranolol completely blocked the effects of epinephrine on cAMP, cAMP-dependent protein kinase, phosphorylase, and glucose-6-P, whereas the alpha blocker phentolamine was totally ineffective. Omission of glucose from the perfusion medium did not modify the effects of epinephrine. Glycogen synthase activity in control perfused and nonperfused muscle was largely glucose-6-P-dependent (-glucose-6-P/+glucose-6-P activity ratios of 0.1 and 0.2, respectively). Epinephrine perfusion caused a small decrease in the enzyme's activity ratio (0.1 to 0.05) and a large increase in its Ka for glucose-6-P (0.3 to 1.5 mM). This increase in glucose-6-P dependency correlated in time with protein kinase activation and was totally blocked by propranolol and unaffected by phentolamine. Comparison of the kinetics of glycogen synthase in extracts of control and epinephrine-perfused muscle with the kinetics of purified rat skeletal muscle glycogen synthase a phosphorylated to various degrees by cAMP-dependent protein kinase indicated that the enzyme was already substantially phosphorylated in control muscle and that epinephrine treatment caused further phosphorylation of synthase, presumably via cAMP-dependent protein kinase. These data provide a basis for speculation about in vivo regulation of the enzyme.

Structural aspects of the membrane-bound Escherichia coli pyridine nucleotide transhydrogenase (EC 1.6.1.1)

Escherichia coli, when grown on complex media containing high levels of amino acids, produce low levels of pyridine nucleotide transhydrogenase (EC 1.6.1 .l) both energy-linked and independent [ 1,2]. Washing and replacement of such cells into a glucose minimal medium resulted in the ‘de nova’ synthesis of both transhydrogenase activities after 3 h [2]. Incorporation of chloramphenicol or omission of glucose in the induction medium resulted in no increase in activity. We have here used this induction system, in which a 5-lo-fold increase in transhydrogenase is produced, to label newly synthesized polypeptides. By incorporating [ 3H] casamino acids in the initial growth phase and non-repressive levels of [ “C]leucine in the induction phase, then partially purifying the transhydrogenase by slight modifications of a described procedure, we have been able to define the synthesized polypeptides on the basis of their high 14(J3H ratios after analysis on polyacrylamide gels in the presence of SDS,

Tension and cycb'c GMP changes in potassium depolarized rabbit colon muscle

Potassium depolarization of rabbit colon muscle elicited a contraction consisting of 2 distinct phases, an initial rapid phasic contraction and a tonic contracture. The tonic contraction was, in contrast to the phasic contraction, dependent on the extracellular calcium for its development. There was a correlation between the tension development and the increase of the cyclic GMP level in the K+-depolarized muscle. Experimental conditions which abolished the tonic contracture, viz glucose omission and treatment with Ca2+-antagonists (verapamil, SKF 525A) also inhibited the cyclic GMP response. The changes of the cyclic GMP levels were Ca2+-dependent. K+-ions also changes the cyclic AMP content an effect which was atropine sensitive. From the experimental data obtained in this investigation we suggest that the co-variation of the tension and the cyclic GMP level in the depolarized colon muscle might depend on oscillations in a common intracellular factor, probably Ca2+.

Control of NAD(P) +-transhydrogenase levels in Escherichia coli

ATP and respiration (NADH)-driven NAD(P) + transhydrogenase (EC 1.6.1.1) activities are low in membranes from Escherichia coli cultured on yeast extract medium (17 and 21 nmol/min × mg) but high on glucose (82 and 142 nmol/min × mg). The ATPase and respiratory activities in both cases appeared comparable. Growth of the bacteria in yeast extract medium followed by washing and replacement into a glucose medium showed that after 3 h the energy-linked and energy-independent NAD(P) + transhydrogenase (reduction of acetylpyridine NAD + by NADPH) activities had appeared simultaneously. Incorporation of chloramphenicol or omission of glucose in the induction medium resulted in no increase in these activities indicating that de novo protein synthesis is required for the induction of energy-linked and -independent NAD(P) + transhydrogenase. It was found that the K m values for acetylpyridine NAD + and NADPH for the energy-independent reaction in membranes from glucose grown cells (143 and 62 μ m) were similar to those in membranes from cells grown on glucose-yeast extract (135 and 45 μ m), respectively, but the maximum velocity at infinite acetyl pyridine NAD + and NADPH increased from 353 to 2175 nmol/min × mg. Furthermore, the membrane-bound NAD(P) + transhydrogenase in glucose-yeast extract grown cells showed substrate inhibition at high NADPH and low acetyl pyridine NAD + levels. Further kinetic data demonstrate that the mechanism of the energy-independent NAD(P) + transhydrogenase in E. coli is similar to that of the mitochondrial enzyme and exhibits similar responses to competitive inhibitors at the NAD + and NADPH sites.

Organic solutes in fluid absorption by renal proximal convoluted tubules.

Proximal convoluted tubules were dissected from rabbit kidneys and perfused with artificial solutions in vitro. The effect of various organic solutes on rate of fluid absorption and transepithelial voltage was tested by removing solutes from or adding them to perfusate and/or bath. Omission of albumin from the bath caused rate of fluid absorption to descrease 33% without any change in voltage. Omission of glucose, lactate, alanine, and citrate from the bath had no effect. In contrast, when they were removed from perfusate, rate of fluid absorption fell by 45-75% (depending on whether they were replaced by NaCl or mannitol and NaCl), and voltage (normally negative in lymen) decreased to near zero. Adding glucose or alanine individually to perfusate caused a small increase in rate of fluid absorption and a relatively large increase in voltage. alpha-Methyl-D-glucoside and cycloleucine (which are transported but not metabolized) had effects similar to glucose and alanine, except that voltage changes were not as great. Phlorizin (10(-5) M in perfusate) had the same effect as removing glucose from perfusate. When glucose and alanine were added to perfusate, epithelial cell swelled significantly. Lactate and citrate also caused rate of fluid absorption to increase when they were added to perfusate, but they did not affect transepithelial voltage nor did they cause cells to swell significantly. Possible mechanisms of these effects and the role of organic solutes in fluid absorption by proximal convoluted tubules are discussed.

Relationships between the exchange of calcium and phosphate in isolated fat-cells.

The uptake and the washout of 45Ca2+ and 32Pi is described in free fat-cells and whole epididymal fat-pads from fed rats. 2. In isolated fat-cells, the uptake of 45Ca2+ proceeds with an initial rapid phase of about 1 min duration, followed by a slower subsequent accumulation. In contrast with the rapid phase, the slow phase is inhibited by 2,4-dinitrophenol, warfarin, oligomycin and verapamil, shows saturation, and presumably represents transport across the plasma membrane. 3. The washout of 45Ca2+ from preloaded cells consists of a rapid (1 min) initial phase and a slow phase which is non-monoexponential, suggesting that the radioactive isotope is released from several cellular pools. 4. When Pi is omitted from the incubation medium, the slow phase of 45Ca uptake is almost abolished, and the washout of 45Ca from preloaded fat-cells is markedly accelerated. At elevated extracellular concentrations of Pi (2,4-6.2mM), the uptake of 45Ca is stimulated by 2-10-fold, and the release of the radioactive isotope from preloaded cells is inhibited. In whole epididymal fat-pads, variations in the extracellular concentration of Pi have no detectable effect on the uptake or the washout of 45Ca. 5. In isolated fat-cells, the accumulation of 32Pi is inhibited by 2,4-dinitrophenol or the omission of glucose from the incubation medium. In a Ca2+-depleted buffer, the uptake of 32Pi is diminished, and hyperosmolarity, which stimulates 45Ca uptake, also accelerates the accumulation of 32Pi. 6. It is concluded that in free fat-cells, the uptake and release of Ca2+ and Pi take place by closely interrelated processes, which are dependent on mitochondrial energy production.

Subcellular distribution of glucocorticoid receptors in mouse fibroblasts

Mouse fibroblasts contain a macromolecular binding component (receptor) which binds glucocorticoids specifically and with high affinity. This study shows that there are three different cellular forms of bound receptor and that it is experimentally possible to markedly alter the subcellular distribution of these three forms. Cells incubated with (3H)triamcinolone acetonide were broken after hypotonic shock and a 7000g hypotonic supernatant was obtained; the pellet was extracted with 0.3 M KCl, yielding a nuclear extract; the remaining pellet was resuspended in water, sonicated, and assayed for "nuclear residual" (i.e., nonextractable) radioactivity. If whole cells are incubated at 0 degrees in a growth medium, almost all of the bound steroid is located in the hypotonic supernatant fraction. Incubation at 37 degrees produces a shift of the steroid-bound macromolecule into the nuclear extractable form, while omission of glucose and addition of KCN at 37 degrees markedly increase the nuclear residual form at the expense of both the nuclear-extractable and supernatant forms. Since DNase treatment of chromatin liberates a soluble steroid-receptor complex, we believe that the nuclear residual form may be steroid-receptor complex tightly bound to chromatin. We propose a model suggesting that an energy-requiring process is required to generate free receptor from the chromatin complex to complete the normal cellular recycling system.

The effect of probenecid on the in vitro absorption of cardiac glycosides

Experiments on the everted sac preparation of the mouse small intestine show that digitoxin absorption and sodium, glucose and water transport are decreased under anaerobic conditions, changed potassium medium contents, omission of glucose and by probenecid, mersalyl and ethacrynic acid. The net absorption of digoxin and ouabain is nearly unchanged in these experiments obviously because a simultaneous increase of the passive diffusion compensates more or less the inhibition of active mechanisms. Therefore the results show that digitoxin absorption is partially mediated by energy resp. oxygen dependent processes. The used methods are not appropriate to demonstrate a similar active transport system for polar glycosides.

Oxidation of Branched Chain Amino Acids by Isolated Hearts and Diaphragms of the Rat: THE EFFECT OF FATTY ACIDS, GLUCOSE, AND PYRUVATE RESPIRATION

Abstract Amino acid oxidation was studied in perfused hearts and incubated hemidiaphragms of rats fed ad libitum. Hearts were perfused with Krebs-Henseleit bicarbonate buffer containing glucose and 0.1 mm amino acids. The addition of 1 mm octanoate to the medium markedly stimulated 14CO2 production from [1-14C]leucine, isoleucine, and valine; inhibited 14CO2 production from [1-14C]alanine, pyruvate, and α-ketoglutarate, and did not affect 14CO2 production from [14C]histidine, threonine, glutamate, ornithine, and phenylalanine. Hemidiaphragms incubated with 1 mm hexanoate or 0.1 to 1.0 mm octanoate increased the oxidation of the branched chain amino acids by 80 to 200% and decreased 14CO2 production from and pyruvate. Octanoate stimulated the oxidation of isoleucine by hemidiaphragms at isoleucine concentrations between 0.01 to 1.0 mm; when isoleucine exceeded 2 mm, octanoate became inhibitory. Octanoate stimulated the oxidation of the branched chain amino acids in the presence or absence of glucose and in the presence or absence of insulin from the medium, in diaphragms and hearts. Bovine serum albumin did not affect branched chain amino acid oxidation in the absence of added fatty acids; it inhibited the stimulatory effect of the latter. The inhibition was proportional to the albumin concentration. Palmitate and oleate (1 mm) added to albumin (15 mg per ml) stimulated branched chain amino acid oxidation by hemidiaphragms; the effect was smaller than that observed with equimolar octanoate and albumin. In hearts, no effect of 1 mm palmitate was observed. Butyrate (2 mm) did not affect branched chain amino acid oxidation by hemidiaphragms; 4 mm dl-β-OH butyrate and 4 mm acetate were inhibitory. In hearts and diaphragms, the omission of glucose from the incubation medium stimulated the oxidation of the branched chain amino acids. Pyruvate added to media containing glucose caused further inhibition of branched chain amino acid oxidation. Insulin stimulated 14CO2 production from [14C]leucine in hearts obtained from rats fasted for 48 hours and perfused without glucose. In hearts perfused with [14C]leucine, the addition of octanoate to the perfusate did not affect the tissue concentration of free leucine nor the labeling of the leucine pool. Octanoate stimulated 14CO2 production from leucine by hemidiaphragms which were preloaded with [14C]leucine, indicating stimulation of leucine oxidation beyond the transport of leucine into muscle cells. The apparent inverse relationship between the oxidation of pyruvate and that of the branched chain amino acids suggests that regulation of the latter may occur at the oxidative decarboxylation of the branched chain α-keto acids in muscles. It is suggested that regulation of branched chain amino acid catabolism in muscles may compliment the alanine cycle, and may play a role in homeostasis under conditions of limited availability of glucose (e.g. fasting) or increased utilization (e.g. exercise).

Production of staphylococcal alpha toxin. II. Glucose repression of toxin formation.

The effect of glucose on alpha toxin production was studied in the Wood 46 strain of Staphylococcus aureus. Optimal toxin production occurred when 0.2% glucose was present in the medium. Omission of glucose gave lower yields of toxin, and concentrations of 0.5% and higher severely depressed toxin formation. Glucose affected the initiation of alpha toxin synthesis in growing cultures. As the glucose concentration increased, the time lag prior to the onset of toxin production also increased, and maximal rates of synthesis were not obtained until essentially all the glucose had been exhausted from the medium. The addition of glucose to toxin-producing cultures caused a temporary, almost complete repression of toxin formation which was not due to pH changes in the culture. The synthesis of most extracellular proteins was not inhibited during the period of repression. After recovery, toxin was produced at rates equal to those of untreated control cultures. The kinetics of toxin repression and the observation that the glucose analogues, 2-deoxy-d-glucose and alpha-methyl-glucoside, as well as other carbon sources, inhibit toxin production suggest that transient repression is responsible for the inhibition of toxin formation. No evidence for a regulatory role of adenosine 3', 5'-cyclc monophosphate in alpha toxin production was obtained.

Effects of isoproterenol on heart muscle performance during myocardial hypoxia

The mechanical performance of isolated rat left ventricular trabecular muscle subjected to hypoxia is improved following the administration of isoproterenol. Enhanced performance, however, is associated with subsequent rapid decline in mechanical activity and the premature appearance of contracture. Glucose omission from the muscle bath accelerates mechanical deterioration of the muscle preparations. A linear relationship between the duration of mechanical activity following the initiation of hypoxia and initial cardiac glycogen concentration is present. For a given glycogen concentration, hypoxia tolerance time is abbreviated in muscles exposed to isoproterenol. Isoproterenol is thus able to enhance heart muscle performance during hypoxia at the expense of early functional deterioration. These effects appear primarily due to isoproterenol-induced accelerated depletion of limited glycolytic reserves of rat heart muscle.

Ethacrynic acid accumulation by renal tissue

During incubation in isotonic glucose-salt medium, thin slices of rabbit kidney tissue accumulate [2- 14C]ethacrynic acid against a concentration gradient. Saturation does not occur in 60 min of incubation, by which time the accumulation is about 10-fold when expressed as a slice : medium ratio. Electron microscopic examination of tissue integrity revealed that there was some swelling and other associated changes after 60 min of incubation also, and the experiments were thus not carried out for longer periods. [2- 14C]ethacrynic acid uptake was markedly lowered by reduction of incubation temperature, omission of glucose from the medium or anoxia. Addition of the inhibitors of cellular metabolism, 2,4-dinitrophenol or sodium azide, or addition of either probenecid or ouabain, which are known to block transport processes, also resulted in a striking decrease in drug accumulation. These results strongly suggest that a metabolically dependent active transport process is involved in the accumulation in vitro of ethacrynic acid by thin slices of rabbit kidney tissue. When slices of rabbit liver tissue were substituted in these experiments, the degree of [2- 14C]ethacrynic acid uptake was greatly reduced and may represent only nonspecific binding to tissue slices. It is suggested that the capacity of renal tissue to accumulate ethacrynic acid in vitro may reflect a process which in vitro enables significant concentration of this drug at pharmacological receptor sites within renal cells. Thus, observed plasma levels of this drug in whole animals may not represent effective pharmacological concentrations.

Effects of deprival of glucose or individual amino acids on polyribosome distribution and rate of protein synthesis in cultured mammalian cells

Using Ehrlich ascites tumor cells in culture, we have examined the effects of omitting glucose or 13 essential amino acids, individually and in combination, on the rate of protein synthesis and the proportions of monomeric and polyribosomal ribosomes in the cells. These data have been used to determine the rate of peptide chain elongation, expressed as the synthesis rate per unit of polyribosomal ribosomes, and to infer a slowing of peptide chain initiation, relative to elongation, from an increase in the proportion of monomeric ribosomes. With omission of glucose from the culture medium, or addition of 2-deoxyglucose, the rates of peptide chain initiation and elongation decreased. These changes, which were rapidly reversed by glucose addition, occur in parallel with, and presumably are directly related to, a decrease in the intracellular ATP concentration. Omission of any one of 13 essential amino acids resulted in an immediate decrease of about 30% in the protein synthesis rate per cell, due largely to a decrease in initiation rate. Omitting combinations of two amino acids had about the same effect as the absence of all 13, producing an approx. 45% decrease in the synthesis rate per cell, again largely due to a slowing of initiation. Although amino acid restriction might be expected to exert a direct effect on the rate of both chain initiation and elongation through lowering of the intracellular aminoacyl-tRNA concentration, operation of some other rate-limiting mechanism is suggested by the fact that lack of any of the other 12 essential amino acids was as effective in slowing initiation as a lack of the initiating amino acid methionine. Evidence for such an indirect mechanism acting through limitation of the energy supply is the observation that deprival of amino acids resulted in a prompt and marked decrease in glucose utilization and in the amount of intracellular ATP. Thus, the effects of amino acid restriction on protein synthesis observed here may be mediated, at least in part, through a decrease in the rate of ATP generation.