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Osmotic effects of membrane permeability in a marine bacterium.

When cells of Alteromonas haloplanktis 214 (ATCC 19855) were preloaded with alpha-[(14)C]aminoisobutyric acid or the K(+) in the cells was labeled with (42)K by incubation in a buffered salt solution containing 0.05 M MgSO(4), 0.01 M KCl, and 0.3 M NaCl, the cells retained their radioactivity when resuspended in the same salt solution. When NaCl was omitted from the solution, 80 to 90% of the radioactivity was lost from the cells. Cells suspended at intermediate concentrations of NaCl also lost radioactivity. New steady-state levels of the intracellular solutes were established within 15 s of suspending the cells; the percentage of radioactivity retained at each level decreased proportionately as the osmolality of the NaCl in the suspending solution decreased. With minor variations in effectiveness, MgCl(2), LiCl, and sucrose could substitute for NaCl on an equiosmolal basis for the retention of radioactivity by the cells. KCl, RbCl, and CsCl were appreciably less effective as replacements for NaCl, particularly when their osmolalities in the suspending solutions were low. The amount of alpha-[(14)C]aminoisobutyric acid taken up by the cells at the steady-state level increased to a maximum as the NaCl concentration in the suspending medium increased to 0.3 M. At suboptimal levels of NaCl, either LiCl or sucrose could substitute for NaCl in increasing the steady-state levels. The results obtained indicate that the porosity of the cytoplasmic membrane of this organism is determined by the difference between the osmotic pressure of the cytoplasm and the suspending medium. The lesser effectiveness of K(+), Rb(+), and Cs(+) than Na(+), Li, or Mg(2+) in permitting the retention of solutes by the cells is attributed to the greater penetrability of the hydrated ions of the former group through the dilated pores of a stretched cytoplasmic membrane.

Aminoisobutyric Acids↗

Insulin-like growth factor-I inhibits dexamethasone-induced proteolysis in cultured L6 myotubes through PI3K/Akt/GSK-3beta and PI3K/Akt/mTOR-dependent mechanisms.

We and others reported previously that IGF-I inhibits dexamethasone-induced proteolysis in cultured L6 myotubes. Recent evidence suggests that this effect of IGF-I at least in part reflects PI3K/Akt-mediated inhibition of Foxo transcription factors. The potential role of other mechanisms, downstream of PI3K/Akt, is not well understood. Here we tested the hypothesis that PI3K/Akt-mediated inactivation of GSK-3beta and activation of mTOR contribute to the anabolic effects of IGF-I in dexamethasone-treated myotubes. Cultured L6 myotubes were treated with 1 microM dexamethasone in the absence or presence of 0.1 microg/ml of IGF-I and inhibitors of GSK-3beta and mTOR. Protein degradation was measured by determining the release of trichloroacetic acid soluble radioactivity from myotubes that had been prelabeled with (3)H-tyrosine for 48 h. IGF-I reduced basal protein breakdown rates and completely abolished the dexamethasone-induced increase in myotube proteolysis. These effects of IGF-I were associated with increased phosphorylation of Akt, GSK-3beta, and the mTOR downstream targets p70(S6K) and 4E-BP1. The PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin reversed the anabolic effect of IGF-I in dexamethasone-treated myotubes. In addition, the GSK-3beta inhibitors LiCl and TDZD-8 reduced protein degradation in a similar fashion as IGF-I. Our results suggest that PI3K/Akt-mediated inactivation of GSK-3beta and activation of mTOR contribute to the anabolic effects of IGF-I in dexamethasone-treated myotubes.

Animals↗

The metabolism of high-molecular-weight ribonucleic acid including polyadenylated species, in the developing rat brain.

High-molecular-weight RNA was isolated from rat brain at various times after the intracranial administration of [32P]Pi. The synthesis of 28S and 18S rRNA could be detected within 1h of the injection of the radioactive precursor and appeared to be more pronounced, relative to other high-molecular-weight RNA, in the brains of older rats compared with those of newborn rats. Polyadenylated RNA, representing most mRNA and their precursors, was isolated by chromatography on oligo(dT)-cellulose. The contribution of this polyadenylated RNA to total RNA synthesis was investigated in the cerebral cortex and the phylogenetically older brain stem at different stages in the development of the rats by using a 5h period of labelling as an arbitrary index of transcription. In the brain stem the proportion of labelled polyadenylated RNA comprised 27-30% of the total RNA. The corresponding values for the cortex decreased from 34% in newborn rats to 23% in 40-150-day-old rats. These data indicated that proportionately more polyadenylated RNA is synthesized in the cortex of the newborn than in the adult rat and that there is a progressive decrease in the synthesis of polyadenylated RNA relative to rRNA during development.

Animals↗

Radiation exposure to the patient and operating room personnel during percutaneous nephrolithotomy.

INTRODUCTION: The increased use of fluoroscopy during percutaneous nephrolithotomy (PCNL) places the urologist and operating room personnel at an occupational risk for measurable radiation exposure. We evaluated the degree of radiation exposure received by the patient and operating room personnel at our endourology facility during PCNL. PATIENTS AND METHOD: The incident radiation dose to the patient and the urologist during 50 consecutive PCNL procedures was monitored using lithium fluoride thermo-luminescent dosimeter chips (TLD chips). A hand held radiation survey meter was used to measure the radiation in air at different positions occupied by various operating room personnel. The approximate distances of the various personnel from the X-ray tube were also measured. RESULTS: PCNL was performed upon 35 males and 15 females. The average time for the procedure was 75 minutes (range: 30-150 min). The mean fluoroscopy screening time during the procedure was 6.04 min (range 1.8-12.16 min) with a mean fluoroscopy tube potential of 68 kVp and a mean tube current of 2.76 mA. The mean radiation exposure dose to the patient was 0.56 mSv (SD +/- 0.35), while the mean incident radiation exposure to the finger of the urologist was 0.28 mSv (SD +/- 0.13). CONCLUSION: The various operating room personnel are within safe radiation dose limits during PCNL. Efficient fluoroscopy further reduces the radiation scatter. All occupational personnel should 'achieve as low as reasonably achievable' dose by adhering to good practices.

Adolescent↗

The permeability of the human erythrocyte to sodium and potassium.

Measurements have been made on the permeability of the human erythrocyte to Na and K in vitro, using radioactive tracers to observe the system in the steady state. The average inward K flux is 1.67 m.eq./liter cells hour, and the apparent activation energy is 12,300 +/- 1300 calories/mol. The inward K flux is independent of the external K concentration in the range of concentrations studied (4 to 16 m.eq. K/liter plasma). Rb appears to compete with K for transport into the cell, whereas Na and Li do not. The average inward Na flux is 3.08 +/- 0.57 m.eq. Na/liter cells hour, and the apparent activation energies are 20,200 +/- 2700 calories/mol for inward transport, and 14,900 +/- 3,400 calories/mol for outward transport. The inward Na flux is dependent on the external Na concentration, but not in a linear fashion. Li appears to compete with Na for inward transport, whereas K and Rb do not. An approximate maximum estimate shows that the energy required for cation transport is only 8.8 calories/mol liter cells hour of the 110 calories/mol liter cells hour available from the consumption of glucose. A working hypothesis for the transport of Na and K is presented.

Biological Transport↗

Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs.

1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke-bicarbonate-[U-(14)C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K(+)-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na(+) and Ca(2+) in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K(+), ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na(+)). 2. Stimulation by K(+) of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na(+), but some synthesis and release of radioactive acetylcholine occurred in the absence of Na(+) as well as in the absence of Ca(2+). 3. The Na(+) dependence of K(+)-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na(+) lack, and partly due to the suppression of the activity of the Na(+) pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca(2+) from the incubation medium. It completely blocked K(+) stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K(+), sodium l-glutamate and lack of Ca(2+) on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took place. In the presence of 4mm-KCl it caused some increase in the release of labelled acetylcholine. 7. The barbiturates (Amytal, pentothal), whilst having no significant effects on labelled acetylcholine synthesis in unstimulated brain except at high concentration (1mm), diminished or abolished (at 0.25 or 0.5mm) the enhanced release of acetylcholine, due to high K(+) or lack of Ca(2+). The fall in tissue content of acetylcholine, due to lack of Ca(2+), was diminished or abolished by pentothal (0.25 or 0.5mm) or Amytal (0.25mm).

Acetylcholine↗

Radioassay for serum and red cell folate.

A simple, reliable assay for serum and red cell folate is described. It uses plain untreated liquid or powdered milk, requiring no special handling or purification, as binder. Such milk makes it possible to ignore endogenous serum folate binder, since crude (but not purified) milk contains a factor which releases folate from serum binder. It simplifies counting radioactivity by employing a gamma emitting isotope of pteroylglutamic acid (PGA), namely the 125I-tyramide of PGA. Like the 3H-PGA assay of Givas and Gutcho, it permits the use of stable PGA rather than unstable methyltetrahydrofolic acid (MeTHFA) standards, because it is carried out at pH 9.3, a pH at which milk folate binder is unable to distinguish PGA from MeTHFA, which is the predominat folate in human tissues. The equipment required to do the radioassay is present in most diagnostic chemistry laboratories. Results are essentially identical to the generally accepted Lactobacillus casel microbiologic method of folate assay, except that false low results are not produced in the radioassay by antibiotics, tranquilizers, and chemotherapeutic agents. Three caveats in its use are the relative instability of 125I-PGA as compared to 3H-PGA, the fact that various powdered milks differ widely in folate-binding capacity, and that only about 60 per cent of commercially obtained skim or powdered milk preparations appear to contain the substance which splits folate from serum binder.

Animals↗

The variations in long term TLD measurements of environmental background radiation at locations in southeastern New York state and northern New Jersey.

The variations of the measured dose rate in air should be recognized especially where background radiation is used as a comparative benchmark to assess radiation surveillance and environmental remediation work. In this note, the natural variations of the combined gamma and cosmic-ray background air-dose rate as measured by lithium fluoride thermoluminescence dosimeters are reported. The dosimeters were deployed monthly at locations within 150 km of the Environmental Measurements Laboratory in New York City. Urban and suburban stations were established with simultaneous indoor and outdoor measurements at some locations. Measurements were obtained over 10 to 18 years. The mean air-dose rates from the six outdoor and four indoor stations vary from 50.8 to 123.1 nGy h(-1). The range of the annual dose rates expressed as a percent-difference of the minimum and maximum is 5.3 to 18.0%. Commonly, 1-mo deviations from the long term mean of about +/-10 to +/-25% are observed. An abrupt decrease in the annual dose rate at one of the measurement sites was attributed to a minor relocation of a dosimeter. Structural shielding factors for the first and second floors of a residence are reported. The ground level location of a dosimeter inside another residence apparently resulted in a very high shielding factor. Finally, a gradual decrease of the dose rate at most of the stations is shown to exist (approximately -0.3 nGy h(-1) y(-1) for the outdoor stations). Plausible causes of this trend are briefly discussed.

Air Pollution, Indoor↗

Degradability of dental ceramics.

The degradation of dental ceramics generally occurs because of mechanical forces or chemical attack. The possible physiological side-effects of ceramics are their tendency to abrade opposing dental structures, the emission of radiation from radioactive components, the roughening of their surfaces by chemical attack with a corresponding increase in plaque retention, and the release of potentially unsafe concentrations of elements as a result of abrasion and dissolution. The chemical durability of dental ceramics is excellent. With the exception of the excessive exposure to acidulated fluoride, ammonium bifluoride, or hydrofluoric acid, there is little risk of surface degradation of virtually all current dental ceramics. Extensive exposure to acidulated fluoride is a possible problem for individuals with head and/or neck cancer who have received large doses of radiation. Such fluoride treatment is necessary to minimize tooth demineralization when saliva flow rates have been reduced because of radiation exposure to salivary glands. Porcelain surface stains are also lost occasionally when abraded by prophylaxis pastes and/or acidulated fluoride. In each case, the solutes are usually not ingested. Further research that uses standardized testing procedures is needed on the chemical durability of dental ceramics. Accelerated durability tests are desirable to minimize the time required for such measurements. The influence of chemical durability on surface roughness and the subsequent effect of roughness on wear of the ceramic restorations as well as of opposing structures should also be explored on a standardized basis.

Biodegradation, Environmental↗

Choline permeability in cardiac muscle cells of the cat.

Permeability of the cardiac cell membrane to choline ions was estimated by measuring radioactive choline influx and efflux in cat ventricular muscle. Maximum values for choline influx in 3.5 and 137 mM choline were respectively 0.56 and 9 pmoles/cm(2).sec. In 3.5 mM choline the intracellular choline concentration was raised more than five times above the extracellular concentration after 2 hr of incubation. In 137 mM choline, choline influx corresponded to the combined loss of intracellular Na and K ions. Paper chromatography of muscle extracts indicated that choline was not metabolized to any important degree. The accumulation of intracellular choline rules out the existence of an efficient active pumping mechanism. By measuring simultaneously choline and sucrose exchange, choline efflux was analyzed in an extracellular phase, followed by two intracellular phases: a rapid and a slow one. Efflux corresponding to the rapid phase was estimated at 16-45 pmoles/cm(2).sec in 137 mM choline and at 1.3-3.5 pmoles/cm(2).sec in 3.5 mM choline; efflux in 3.5 mM choline was proportional to the intracellular choline concentration. The absolute figures for unidirectional efflux were much larger than the net influx values. The data are compared to Na and Li exchange in heart cells. Possible mechanisms for explaining the choline behavior in heart muscle are discussed.

Animals↗

[3H]Ethylpropylamiloride, a ligand to analyze the properties of the Na+/H+ exchange system in the membranes of normal and hypertrophied kidneys.

[3H]Ethylpropylamiloride is a useful radioactive label to identify the Na+/H+ exchange system (Vigne, P., Frelin, C., Audinot, M., Borsotto, M., Cragoe, E. J., and Lazdunski, M. (1984) EMBO J. 3, 2647-2651). This paper extends the analysis of the properties of interaction of [3H]ethylpropylamiloride with the exchanger and describes its use with hypertrophied kidneys. [3H]Ethylpropylamiloride-binding sites copurify with the luminal membrane marker alkaline phosphatase but not with the basolateral membrane marker (Na+,K+)ATPase, thus indicating an asymmetric distribution of the Na+/H+ exchanger. Specific [3H]ethylpropylamiloride binding is dependent on pH. The pH dependency indicates that an ionizable function with a pKapp of 7.0 is essential in the association of the amiloride derivative. H+ acts competitively on [3H]ethylpropylamiloride binding; Na+, Li+, or cholinium ions have no effect on the association. Compensatory adaptation of the kidney to chronic reduction of renal mass is accompanied by a 1.7-fold increase in the activity of the Na+/H+ exchange system. Properties of interaction of internal and external pH with the Na+/H+ exchanger of normal and hypertrophied kidneys are identical. Titration of [3H]ethylpropylamiloride-binding sites in normal and hypertrophied kidneys suggests that the increased activity of the Na+/H+ exchange system is not accompanied by an increased concentration of exchangers.

Amiloride↗

Inhibition of Na+/H+ exchange in the rat is associated with decreased ursodeoxycholate hypercholeresis, decreased secretion of unconjugated urodeoxycholate, and increased ursodeoxycholate glucuronidation.

In the perfused rat liver, ursodeoxycholate in high dose produces an HCO3- -rich hypercholeresis which we have shown previously to be inhibited by replacement of perfusate Na+ with Li+ or by addition of amiloride (or amiloride analogues). In the present studies, we have determined whether such inhibition is associated with altered ursodeoxycholate biotransformation. Under control conditions, ursodeoxycholate infusion produced a 3.7-fold increase in bile flow and a 9.2-fold increase in biliary HCO3- output. By thin-layer chromatography, ursodeoxycholate radioactivity in bile was present in unconjugated form (15%) or as glycine or taurine amidates. Glucuronide conjugates of ursodeoxycholate accounted for less than 1% of biliary bile acids. Li+/Na+ substitution decreased ursodeoxycholate-stimulated bile flow and HCO3- secretion by greater than 90%, but decreased recovery of ursodeoxycholate and metabolites by only 25%. Amiloride or amiloride analogues decreased ursodeoxycholate-stimulated choleresis and HCO3- output by 38%-76%, yet did not cause decreased recovery of ursodeoxycholate and metabolites. Inhibition of the hypercholeresis was associated with a decrease in unconjugated ursodeoxycholate to less than 2% of total biliary bile acids, a striking increase in ursodeoxycholate glucuronides, and a reciprocal decrease in glycine and taurine amidates. With Li+/Na+ substitution, the predominant metabolites were a mixture of the 24-ester and the 3-aketal (ethereal) glucuronide (29%), and amidation with glycine appeared to be selectively inhibited; with amiloride or its analogues, only the 3-ethereal glucuronide was formed (20%-60% of biliary bile acids), and both taurine and glycine amidation were inhibited. Thus, maneuvers that decrease Na+/H+ exchange inhibit ursodeoxycholate hypercholeresis and cause replacement of unconjugated ursodeoxycholate in bile by its glucuronide. The secretion of unconjugated ursodeoxycholate, a lipophilic bile acid, appears to be necessary for hypercholeresis induced by high-dose ursodeoxycholate infusion.

Amiloride↗

(-)-N-[(11)C]propyl-norapomorphine: a positron-labeled dopamine agonist for PET imaging of D(2) receptors.

Imaging neuroreceptors with radiolabeled agonists might provide valuable information on the in vivo agonist affinity states of receptors of interest. We report here the radiosynthesis, biodistribution in rodents, and imaging studies in baboons of [(11)C]-labeled (-)-N-propyl-norapomorphine [(-)-NPA]. (-)-[(11)C]NPA was prepared by reacting norapomorphine with [(11)C]propionyl chloride and a lithium aluminum hydride reduction. [(11)C]Propionyl chloride was prepared by reacting [(11)C]CO(2) with ethylmagnesium bromide, followed by reacting with phthaloyl chloride. The radiochemical yield of (-)-[(11)C]NPA was 2.5% at end of synthesis (EOS), and the synthesis time was 60 min. The specific activity was 1700+/-1900 mCi/micromol ( N=7; ranged 110-5200 mCi/micromol at EOS). Rodent biodistribution studies showed high uptake of [(11)C](-)-NPA in D(2) receptor-rich areas, and the striatum/cerebellum ratios were 1.7, 3.4, and 4.4 at 5 min, 30 min, and 60 min postinjection, respectively. Pretreating the animals with haloperidol (1 mg/kg) decreased the striatum/cerebellum ratio at 30 min postinjection to 1.3. (-)-[(11)C]NPA was also evaluated via baboon positron emission tomography (PET) studies. Under control conditions ( N=4), rapid uptake of the tracer was observed and the striatum/cerebellum ratio reached 2.86+/-0.15 at 45 min postinjection. Following haloperidol pretreatment (0.2 mg/kg IV), the striatum/cerebellum ratio was 1.29 at 45 min postinjection. The result demonstrated the existence of specific binding of this new tracer to the D(2) receptor. To our knowledge, the current finding of a striatum/cerebellum ratio of 2.8 in baboon was the highest reported with a radiolabeled D(2) agonist. (-)-[(11)C]NPA is a promising new D(2) agonist PET tracer for probing D(2) receptors in vivo using PET.

Animals↗

Sodium-dependent influx of orthophosphate in mammalian non-myelinated nerve.

1. The rate of uptake of radiophosphate was measured in desheathed vagus nerves of rabbits mounted in an apparatus where the incubating solution flowed along the preparation. 2. Replacement of the Na of the Locke by either choline or Tris slowed the rate of uptake to about 10% of its value in Na; with K it was slowed to 20%; and with Li to 50%. 3. Measurements of the rate of uptake at different extracellular phosphate concentrations showed that in choline-Locke the influx of phosphate was proportional to the extracellular phosphate concentration, while in Locke the rate of inward flow showed a tendency to saturation with increasing phosphate concentrations. 4. Extracts of nerves after 45 min exposure to labelled solutions showed for different phosphate concentrations a slowing of the labelling of ATP, ADP and creatine-phosphate (CrP) and inorganic phosphate (Pi) when Locke was replaced by choline-Locke. 5. A similar slowing was found when, at 0-2 mM phosphate, the labelling of these compounds was measured at different times: application of choline-Locke reduced the rate of incorporation of 32P to about 15% of the rate in Locke. 6. In preparations that were loaded with radiophosphate and then washed with inactive solution, and the effluent fractionated, over 90% of the radioactivity was found in the inorganic phosphate fraction. 7. The efflux of 32P showed an initial exponential phase with a time constant of 20-30 min and a much slower one. The slow efflux had a rate constant of 0-0014 min-1 in 0-2 mM external phosphate. 8. Increasing the external phosphate concentration increased the efflux. 9. Prolonged incubation in choline-Locke reduced the efflux. 10. The influx of phosphate was calculated for different external phosphate concentrations using the rate of uptake of radiophosphate measured 45 min after the application of isotope and the corresponding efflux rate coefficient and the intracellular specific activities of the labelled compounds. 11. A correction was also introduced for diffusion, using the "limited biophase model". 12. The Na-sensitive influx, i.e. the difference between influx in Locke and influx in choline-Locke, showed saturation kinetics. 13. A Lineweaver-Burk plot for Na-sensitive uncorrected influxes gave a vmax of 16-7 mumole/kg wet wt. min and an apparent Km of 0-42 mM; for the corrected fluxes these values were 18-2 and 0-36. 14. It is concluded that a large part of phosphate influx and some of the phosphate efflux is mediated by a specific Na-dependent phosphate transport system. 15. This system seems to be present also in other types of nervous tissues and probably in many types of animal cells.

Adenosine Diphosphate↗

The dependence of calcium efflux from cardiac muscle on temperature and external ion composition.

1. Exchangeable Ca in guinea-pig auricles and ventricular trabeculae of sheep and calf hearts was labelled with (45)Ca and the loss of radioactivity into inactive rinsing solutions of different ion composition was measured for periods up to 6 hr. At no time did the decrease of radioactivity in the muscle follow a single exponential course, while the rate coefficient k (= fraction of (45)Ca lost per minute from muscle into rinsing solution) decreased slightly with time.2. On the basis of the temperature-sensitivity of Ca efflux from auricles the activation energy has been calculated to have a value of 5900 cal/mole, corresponding to a Q(10) of 1.35. 2,4-Dinitrophenol (5.5 x 10(-5) - 5.5 x 10(-4)M) had either no effect on Ca efflux or increased it slightly.3. Compared to control efflux in 1.8 mM [Ca](o) Ca efflux decreased to 70% in Ca-free solution, to 20% in Ca-free, Na-free solution and to 65% in Ca-containing, Na-free solution, NaCl being replaced by either sucrose or LiCl. Quantitatively, Ca efflux from auricles has been shown to depend to a large extent on the ratio [Ca(2+)](o)/[Na(+)](o) (2). The affinity for Na of the activation site for Ca efflux (carrier) is much less than for Ca.4. The efflux from muscles soaked for about 2 hr in Ca-free solution was not linearly related to Ca-concentration in the tissue but followed a square law.5. While Ca content in auricles increased in Ca-containing, Na-poor solution it decreased again when Tyrode solution was readmitted indicating a Na-sensitive Ca net transport in cardiac muscle.6. The results are interpreted in terms of a modified exchange diffusion mechanism (Ussing, 1947) which is responsible for Ca extrusion from mammalian cardiac muscle.

Animals↗

Cation coupling to melibiose transport in Salmonella typhimurium.

Melibiose transport in Salmonella typhimurium was investigated. Radioactive melibiose was prepared and the melibiose transport system was characterized. Na+ and Li+ stimulated transport of melibiose by lowering the Km value without affecting the Vmax value; Km values were 0.50 mM in the absence of Na+ or Li+ and 0.12 mM in the presence of 10 mM NaCl or 10 mM LiCl. The Vmax value was 140 nmol/min per mg of protein. Melibiose was a much more effective substrate than methyl-beta-thiogalactoside. An Na+-melibiose cotransport mechanism was suggested by three types of experiments. First, the influx of Na+ induced by melibiose influx was observed with melibiose-induced cells. Second, the efflux of H+ induced by melibiose influx was observed only in the presence of Na+ or Li+, demonstrating the absence of H+-melibiose cotransport. Third, either an artificially imposed Na+ gradient or membrane potential could drive melibiose uptake in cells. Formation of an Na+ gradient in S. typhimurium was shown to be coupled to H+ by three methods. First, uncoupler-sensitive extrusion of Na+ was energized by respiration or glycolysis. Second, efflux of H+ induced by Na+ influx was detected. Third, a change in the pH gradient was elicited by imposing an Na+ gradient in energized membrane vesicles. Thus, it is concluded that the mechanism for Na+ extrusion is an Na+/H+ antiport. The Na+/H+ antiporter is a transformer which converts an electrochemical H+ gradient to an Na+ gradient, which then drives melibiose transport. Li+ was inhibitory for the growth of cells when melibiose was the sole carbon source, even though Li+ stimulated melibiose transport. This suggests that high intracellular Li+ may be harmful.

Biological Transport↗

Agonist-mediated formation of inositol monophosphate isomers in rat cortical prisms.

The carbachol and adrenaline-mediated accumulation of inositol monophosphate isomers in rat cortical prisms has been studied using a commonly employed experimental protocol involving preincubation with myo-[2-3H]-inositol and subsequent incubation with agonists in the presence of 10 mM LiCl. Inositol phosphate isomers have been analysed by HPLC and identified by comparison of their elution characteristics with those of commercially available standards and the degradation products of authentic Ins 1,3,4-P3 and Ins 1,4,5-P3. Incubation of prelabelled cortical prisms for 1 hr with 10 mM LiCl alone gives rise to accumulation of radioactivity in two inositol monophosphate peaks which co-elute with Ins 1-P and Ins 4-P and one major bisphosphate peak which co-migrates with Ins 1,4-P2. Most of the monophosphate radioactivity is recovered in the Ins 4-P peak (Ins 1-P/Ins 4-P labelling ratio 0.68). Both carbachol and adrenaline produce dose-dependent increases in the labelling of Ins 1-P and Ins 4-P which are antagonized by atropine and prazosin respectively. However, carbachol produces a larger stimulation of accumulation of both monophosphates and also gives rise to a larger selective increase in the accumulation of Ins 1-P (Ins 1-P/4-P labelling ratio 1.40 in the presence of 1 mM carbachol, 0.98 in the presence of 1 mM adrenaline). Kinetic studies of the carbachol-stimulated increases in inositol mono- and bisphosphate labelling have revealed that, in the early period following carbachol addition (0-5 min), Ins 4-P and Ins 1,4-P2 are labelled more rapidly than Ins 1-P, whereas the reverse is true at later periods (15-60 min) of the incubations. These observations, coupled with the low levels of labelling of the major Ins 1,3,4-P3 breakdown products (Ins 1,3-P2 and Ins 3,4-P2) compared with that of Ins 1,4-P2, suggest that large-scale production of Ins 1-P is a comparatively late feature of carbachol-mediated inositol phospholipid metabolism and that, if the Ins 1-P is derived from breakdown of Ins 1,3,4,5-P4 via Ins 1,3,4-P3, the turnover of Ins 1,3-P2 + Ins 3,4-P2 must be approximately one order of magnitude greater than that of the Ins 4-P precursor, Ins 1,4-P2.

Animals↗

Glycine fluxes in squid giant axons.

1. The influx of a number of amino acids into squid giant axons has been studied. Particular emphasis has been placed on glycine and to a lesser extent glutamate. 2. To facilitate the study of the uptake of 14C-labelled amino acids a technique was devised in which the 14C taken up was measured directly in the intact axon with a glass scintillator fibre. This technique gave results similar to the usual technique in which the axoplasm was extruded for the assay of radioactivity. 3. The changes in glycine influx with extracellular glycine concentration suggests that two saturating components are present, one with high affinity and one with low affinity. 4. The glycine influx does not seem normally to be sensitive to the removal of extracellular sodium by replacement with choline. A Na-sensitive component appeared, however, after a period of immersion in artificial sea water. There was also some depression of glycine influx if Na were replaced by Li. 5. Glutamate uptake was greatly reduced by removal of extracellular Na in confirmation of work by Baker & Potashner (1973). Orthophosphate uptake was also greatly reduced by removal of extracellular Na. 6. CN reversibly inhibited glycine uptake after a delay, indicating that part of the uptake mechanism may require ATP. 7. 14C-labelled glycine injected into squid axons was found not to exchange to any serious extent with other compounds over periods of a few hours. The glycine efflux could therefore be studied. This was found to be markedly increased by extracellular glycine and by certain other neutral amino acids applied extracellularly in the artificial sea water. 8. The enhanced glycine efflux in extracellular glycine was not affected by ouabain and CN. 9. It is suggested that glycine uptake in squid axons involves two components. One is sensitive to CN and ouabain and probably derives energy from ATP break-down. The other is probably an ATP independent exchange diffusion system in which other amino acids as well as glycine can exchange for glycine. Both these systems are independent of extracellular Na concentration. A third Na-dependent system may appear under certain conditions.

Amino Acids↗