New technique for measuring dynamic axonal transport and its application to temperature effects

A new technique was devised for the dynamic detection of the axoplasmic transport of β-radioactively labeled materials in which a semiconductor radiation detector was used as the β-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the β-radioactive distribution of axoplasmic transport could be measured in an axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 °C. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 °C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q₁₀ is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. This technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

Fast axonal transport in motor nerve regeneration

This report describes the fast axonal transport of [³H]-leucine-labeled proteins in regenerating rat sciatic motor nerves. A normal rate of fast transport (383 ± 33 mm/day) was present in the regenerating sprouts, as well as in the central stumps. The rapidly transported proteins passed the level of axotomy without impediment, and accumulated in the endings of the regenerating sprouts, as shown by electron microscope autoradiography. In addition, transported proteins accumulated in terminal neuromas. The relative amount of protein-incorporated radioactivity in the crest of fast transport in the regenerating nerves was increased compared to control nerves. These results are interpreted to suggest that the mechanism of fast transport is the same in regenerating sprouts as in normal axons; during regeneration fast transport appears to add newly synthesized materials to the growing tip.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.     

Continuous monitoring of rhizosphere respiration after labelling of plant shoots with 14CO2

The present work describes an original method to follow rate of ¹⁴CO₂ and total CO₂ production from rhizosphere respiration after plant shoots had been pulse-labelled with ¹⁴CO₂. We used a radioactivity detector equipped with a plastic cell for flow detection of beta radiation by solid scintillation counting. The radioactivity detector was coupled with an infrared gas analyser. The flow detection of ¹⁴CO₂ was compared to trapping of ¹⁴CO₂ in NaOH and counting by liquid scintillation. First, we demonstrated that NaOH (1 M) trapped 95% of the CO₂ of a gaseous sample. Then, we determined that the counting efficiency of the radioactivity flow cell was 41% of the activity of gaseous samples as determined by trapping in NaOH (1 M) and by counting by static liquid scintillation. The sensitivity of the ¹⁴CO₂- flow detection was 0.08 Bq mL⁻¹ air and the precision was 2.9% of the activity measured compared to 0.9% for NaOH trapping method. We presented two applications which illustrate the relevance of ¹⁴CO₂-flow detection to investigations using 14C to trace photoassimilates within the plant-soil system. First, we examined the kinetics of 14CO₂ production when concentrated acid is added to NaH¹⁴CO₃. This method is the most commonly used to label photoassimilates with ¹⁴C. Then, we monitored ¹⁴CO₂ activity in rhizosphere respiration of 5-week old maize cultivated in soil and whose shoots had been pulse-labelled with ¹⁴CO₂. We conclude that alkali traps should be used for a cumulative determination of ¹⁴CO₂ because they are cheap and accurate. On the other hand, we demonstrated that the flow detection of ¹⁴CO₂ had a finer temporal resolution and was consequently a relevant tool to study C dynamics in the rhizosphere at a short time scale. This revised version was published online in June 2006 with corrections to the Cover Date.     

DIVALENT CATION SPECIFICITY OF THE CALCIUM REQUIREMENT FOR FAST TRANSPORT OF PROTEINS IN AXONS OF DESHEATHED NERVES

The presence of a requirement for calcium during the fast transport of [³H]protein in axons was assessed in desheathed spinal nerves of bullfrog. The nerves were desheathed locally along 4 mm of their length, and desheathing was judged effective on the basis of an enhanced uptake of [³H]leucine into that region of nerve trunk. Desheathing per se had a slight inhibitory effect on transport. Incubation of desheathed nerve trunks in calcium-free medium reduced transport by 60-80% relative to that in desheathed nerves incubated in normal medium. Addition of Mg²⁺ or Sr²⁺ to the calcium-free medium allowed transport to proceed normally. Addition of Co²⁺ or Mn²⁺ to normal medium did not affect transport in desheathed isolated nerve trunks. When ganglia and nerve trunks were both incubated in medium containing 0.18 mM-CoCl₂, transport was depressed to a similar extent proximal and distal to the desheathed region. This again indicates that Co²⁺ does not inhibit transport in desheathed nerves, whereas it does inhibit transport in the ganglia. Additive inhibitory effects were observed when ganglia were incubated in medium containing 0.018 mM-CoCl₂, and desheathed nerves were incubated in calcium-free medium. Differences in the divalent cation specificities of the axonal and ganglionic calcium requirements suggest that calcium supports transport in nerves in a manner distinct from its role in maintaining transport in spinal ganglia. It is concluded that the ganglionic calcium requirement involves initiation of axonal transport in the soma rather than translocation in the intraganglionic region of axon.     

Axonal transport of muscarinic receptors in vesicles containing noradrenaline and dopamine-β-hydroxylase

Presynaptic muscarinic receptors labeled with [³H]dexetimide and noradrenaline in dog splenic nerves accumulated proximally to a ligature at the same rate of axonal transport. After fractionation by differential centrifugation, specific [³H]quinuclidinyl benzilate or [³H]dexetimide binding revealed a distribution profile similar to that of dopamine-β-hydroxylase and noradrenaline. Subfractionation by density gradient centrifugation showed two peaks of muscarinic receptors; the peak of density 1.17 contained noradrenaline and dopamine-β-hydroxylase whereas that of density 1.14 was devoid of noradrenaline. Therefore the foregoing experiments provide evidence that presynaptic muscarinic receptors are transported in sympathetic nerves in synaptic vesicles which are similar to those containing noradrenaline and dopamine-β-hydroxylase. This suggests a possible coexistence of receptor and neurotransmitter in the same vesicle.     

New technique for measuring dynamic axonal transport and its application to temperature effects.

A new technique was devised for the dynamic detection of the axoplasmic transport of beta-radioactively labeled materials in which a semiconductor radiation detector was used as the beta-ray counter. The detector element is a silicon p-n junction diode and has a diameter of 2.0 mm. With this detector, the beta-radioactive distribution of axoplasmic transport could be measured in a axon maintained physiologically without cutting nerves. This method makes possible determination of the transport rate using one bundle of peripheral nerves. The rate in the bullfrog was 6.4 mm per hour at 24.0 degrees D. Temperature effects on the bullfrog axoplasmic transport were also observed at different temperatures, ranging from 5.0 to 24.0 degrees C. At these temperatures the rate increased as an exponential function of temperature from 1.1 to 6.4 mm per hour. Within this temperature range, the Q10 is 2.5 and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 Kcal. this technique offers great advantages in permitting direct study of the axoplasmic flow of the axon in a physiological condition.     

Direct counting of tissues containing radioactive cholesterol by a two-phase liquid scintillation method

A rapid and simple method for counting radioactivity in tissue samples containing [³H]- or [¹⁴C]-cholesterol is described.Up to 500 μl of the specimen to be counted (plasma, tissue homogenate) is measured into a counting vial. The lipids of the tissue are extracted into 15 ml of a toluene-based scintillation mixture containing 37.5% ethylene glycol monomethyl ether that is added to the same vial. With the addition of 1 ml water, two phases form: the upper toluene phase containing all cholesterol together with the scintillating phosphor and the lower water phase containing most of the quenching material. Bleaching to reduce color quenching is not necessary. Chemiluminescence is negligible. The counting efficiencies are appreciably higher than those obtained in aqueous one-phase scintillation systems but lower than those obtained with pure standards in one-phase pure toluene scintillation systems.     

Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5)

The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na⁺-glutamine_ex/glutamine_in transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K⁺ gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na⁺. Internal Na⁺ exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.     

IS THERE BIDIRECTIONAL TRANSPORT OF NORADRENALINE IN SYMPATHETIC NERVES?

The experiments were designed to detect somatopetal transport of [¹⁴C]noradrenaline in the postganglionic sympathetic nerves supplying the cat spleen and sheep eye. The animals were treated with nialamide to protect the radioactive noradrenaline, after uptake into the nerve terminals, from monoamine oxidase. In the spleen, the transmitter stores were labelled by infusion of [¹⁴C]noradrenaline into a branch of the splenic artery. The branches of the nerves to the infused and non-infused sides of the spleen were ligated in an attempt to arrest, distal to the constriction, any noradrenaline transported somatopetally in the axons from their terminals. After 24 hr, however, there was less radioactivity in the nerves distal compared to proximal to the constriction, despite heavier labelling of the terminal transmitter stores in the infused portion of the spleen. The proximal accumulation of radioactivity could be attributed to a somatofugal transport of [¹⁴C]noradrenaline. Experiments were also done on the intact sympathetic nerve supply of the sheep eye. The sympathetic nerve terminals in the smooth muscle of the left eye were heavily labelled 5 days after the injection of [¹⁴C]noradrenaline into the left vitreous humour. However, both superior cervical ganglia were only lightly labelled, and there was no significant difference in the radioactivity present in the two ganglia. The results provide no support for a bidirectional transport of noradrenaline in sympathetic nerves but are consistent with a somatofugal transport of the amine storage vesicles from their site of synthesis in the soma to the axon terminals.     

A new method to evaluate the residual activity in patients undergoing 131I thyroid therapy

The radioiodine administration is a standard therapeutic approach to both benign thyroid diseases, such as hyperthyroidism, and carcinomas. The high administered ¹³¹I activities are of radiation protection concern, due to relevant patient residual contamination. The aim of this work was to develop a new procedure based on external radiometric surveys and on a mathematical model in order to estimate the ¹³¹I activity in patients undergoing hyperthyroidism radioiodine therapy.In the first stage of this study, a suitable detector was chosen and its response vs. activity was characterized. The experimental verification was performed measuring the ambient dose equivalent rate from patients receiving radioiodine administration. The results confirm the reliability of the proposed method, as shown by the slight differences between the administered activities and the ones calculated from external measurements. Furthermore, the same procedure was applied to detect the percentage residual activity in patients at two preset time intervals: 4 hours and 4 days after the radioiodine administration. The obtained results clearly highlight that the method can ensure a level of reliability compatible with the radiation protection purposes.     

The surfactant Bio-Solv-BBS-3 as a scintillator in liquid scintillation counting

When the surfactant mixture Bio-Solv-BBS-3 is added to a scintillation solvent it acts as a primary scintillator in response to β emissions (and Compton electrons from γs). The fluorescence excitation threshold is higher and fluorescence yield is lower than those of the primary scintillators usually employed in scintillation counting. Presence of a surfactant in a sample containing ¹⁴C or more energetic βs will be counted at higher efficiency than would be indicated by a quench correction curve (efficiency vs sample channels ratios or external standard channels ratios) derived from standards not containing surfactant.     

Equilibrium isoelectric focussing in acrylamide gel slabs--reduction of cathodic drift.

A simple, economical method for counting acrylamide gel slices on solid filter paper supports in a toluene-based scintillation cocktail is described. Major advantages of the system include no requirement for either dissolution of the gel or elution of the radioactive material prior to emulsion counting and the direct reutilization of scintillation cocktail and vials. Additionally, ³²P-labeled RNA samples can be counted with better relative efficiencies and those labeled with ¹⁴C or ³³P can be determined at equivalent efficiencies. Tritium was detected less readily, with an absolute efficiency of approximately 10%.     

Inhibition of fast axonal transport in vitro by the local anesthetics prilocaine, mepivacaine, and bupivacaine

The aim of the present study was to establish the concentrations of prilocaine, mepivacaine, and bupivacaine which are effective at blocking fast axonal transport, to determine whether prilocaine and mepivacaine offer a better prospect of dissociating conduction block and transport block in vivo than does lidocaine and whether bupivacaine offers a better prospect than etidocaine in the same context. Fast axonal transport of [3H]leucine-labeled proteins was studied in vitro in bullfrog spinal nerves and quantitated by liquid scintillation counting. Exposure of spinal nerves to 14 mM prilocaine reduced the quantity of 3H-labeled proteins which accumulated at a ligature by 86%, and exposure to 14 mM mepivacaine reduced it by 70%; 10 mM prilocaine reduced this same parameter by 54%, a degree of inhibition close to the 44% reduction caused by 14 mM lidocaine. The D(-) and L(+) stereoisomers of mepivacaine each reduced transport to the ligature by approximately 50% at a concentration of 14 mM. Bupivacaine reduced the accumulation of 3H-labeled proteins at the ligature by 49% at a 10 mM concentration (pH 6.2); its potency is close to that found for etidocaine in a previous study. Since prilocaine and mepivacaine are at least as potent as lidocaine as transport inhibitors and at blocking impulse conduction, these two anesthetics offer no advantage over lidocaine to achieve dissociation of conduction block from transport block in vivo. Bupivacaine appears to offer no advantage over etidocaine in the same context, as the two agents have a similar potency as local anesthetics and a similar potency as inhibitors of fast axonal transport.     

Application and efficiency of scintillation autoradiography for Drosophila polytene chromosomes

Summary A rapid method of autoradiography using the scintillation cocktail (Toluene and scintillation fluid, Omnifluor) has been described earlier. Its application and efficiency have been tested using both ³H-thymidine and ³H-uridine. The optimum time required for processing the autoradiograms has been found to be 24 h dry exposure followed by 48 h in the scintillation mixture. Detailed analysis of the autoradiograms with ³H-uridine reveals that with the rapid method the 100% level of labelling index is reached by 48 h while with the conventional method the same level is reached by 10 to 12 days of dry exposure. The maximum grain density is reached by 16 to 17 days by the conventional method. While by the rapid method, the maximum grain density is approximately 80% of the control, this grain density is reached by 48 h (plus 24 h of dry exposure) and thereafter forms a plateau. With Toluene alone the grain density never exceeds 20%. The background is also relatively low and less variable in the O-T-processed autoradiograms, as compared to the two controls. These results support that the scintillation fluid plays the key role in augmenting the labelling. Furthermore, although the maximum grain density by the rapid technique is 80% of the control, the grain density obtained by the rapid method gives less coincidence and superimposition of grains.On the other hand, with ³H-thymidine, although all labelling patterns could be resolved, the labelling index (i.e., percent of labelled cells) is about 40% at 48 h (plus 24 h) and about 79.5% at 96 h with the rapid method, as compared to about 30% and 44% with the conventional method at the two time points, respectively. Only with 16–17 days' dry exposure the ³H-thymidine labelling index increases to 67%. The frequency of the initial patterns (DD-2C) which are usually less frequent, has been found to have increased with the rapid method. No difference in grain density of labelling of ³H-thymidine could be detected between the rapid method and the conventional method. The resolution of grains also seems to be better by the rapid method, due probably to smaller size and lack of superimposition of grains. Other applications, advantages and limitations have been discussed.     

Improvements in and Environmental Applications of Double-Vial Radiorespirometry for the Study of Microbial Mineralization

Several variations in the scintillation mixture and the filter paper arrangements for double-vial radiorespirometry were compared. Improved efficiencies (44%) and shorter response times were found by adding wetting agents and methanolic NaOH to the scintillation mixture in the filter paper. The scintillation chemicals used did not contain dioxane and were found to be nontoxic to the test microbiota in this system. Covering the inner reaction vial with aluminum foil minimized the reduction in counting efficiency when testing colored or dense environmental samples. Mineralization rates were determined with ¹⁴C-labeled glucose, acetate, and glutamate and [¹⁴C]cellulose- and [¹⁴C]lignin-labeled lignocellulose for composting cow manure, forest soil, and arctic lake sediment microbiota. This improved method can be used in a variety of procedures involving the measurement of microbial mineralizations of organic compounds. Since no liquid scintillation cocktail is used for counting, the radioactive wastes are aqueous or can be incinerated, making disposal easy.     

Involvement of intracellular calcium in the phosphate efflux from mammalian nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from desheathed rabbit vagus nerves after loading with radiophosphate. The effects of strategies designed to increase intracellular calcium were investigated. At the same time, the exchangeable calcium content was measured using⁴⁵Ca. Application of calcium ionophore A23187 increased phosphate efflux in the presence of external calcium in parallel with an increase in calcium content. In the absence of external calcium, there was only a late, small increase in phosphate efflux. For nerves already treated with the calcium ionophore, the phosphate efflux was sensitive to small changes in external calcium, in the range 0.2 to 2mm calcium, whereas similar increases in calcium in absence of ionophore gave much smaller increases in phosphate efflux. Removal of external sodium (choline substitution) produced an initial increase in phosphate efflux followed by a fall. The initial increase in phosphate efflux was much larger in the presence of calcium, than in its absence. The difference was again paralleled by an increase in calcium content of the preparation, thought to be due to inhibition of Na/Ca exchange by removal of external sodium. Measurements of ATP content and ATP, ADP, phosphate and creatine phosphate ratios did not indicate significant metabolic changes when the calcium content was increased. Stimulation of phosphate efflux by an increase in intracellular calcium may be due to stimulation of phospholipid metabolism. Alternatively, it is suggested that stimulation of phosphate efflux is associated with the stimulation of calcium efflux, possibly by cotransport of calcium and phosphate.     

Semiquantitative estimations of quinacrine fluorescence in intestinal nerve fibres

Summary Quinacrine has been shown to bind selectively to a population of nerves and ganglion cells in the mouse, rat and guinea-pig gut. In the present report a method for semiquantitation of nervous quinacrine contents using semiquantitative estimations of fluorescence intensity and nerve fibre density is presented and evaluated. Estimation of fluorescence intensity and nerve fibre density is based on a scale with eleven steps from 0 to 5. Preparations were incubated in ¹⁴C-labelled quinacrine hydrochloride. Reliability of the scale was expressed as the correlation coefficient between two consecutive blind estimations of the same preparations with recoding and remixing specimens in between. This correlation was found to be 0.95 or higher. Validity was expressed as the correlation between the semiquantitative estimations and ¹⁴C-quinacrine uptake measurements into the same specimens. Also this correlation was found to be strongly positive.It was concluded that nervous quinacrine content and amount of quinacrine binding nerves can be reliably estimated by fluorescence microscopy.     

Radiolabeled polyvinyl alcohol particles: A potential agent to monitor embolization procedures

Polyvinyl alcohol sponge (PVA) is a widely used angiographic embolic agent. The radiolabeling of PVA can accurately identify particle localization and may decrease the possibility of patient morbidity from embolization to distal sites. We incorporated ^99mTc sulfur colloid (SC) into PVA by heating. Animal experiments demonstrated the in vivo stability of the ^99mTc SC-PVA complex and the efficacy of external imaging. ^99mTc SC-PVA biodistribution data and external NaI(Tl) scintillation probe counts were performed, to assess anatomic localization. Embolization with this complex was performed in a patient.     

Beyond non-integer Hill coefficients: A novel approach to analyzing binding data,applied to Na+-driven transporters

Prokaryotic and eukaryotic Na⁺-driven transporters couple the movement of one or more Na⁺ ions down their electrochemical gradient to the active transport of a variety of solutes. When more than one Na⁺ is involved, Na⁺-binding data are usually analyzed using the Hill equation with a non-integer exponent n. The results of this analysis are an overall K_d-like constant equal to the concentration of ligand that produces half saturation and n, a measure of cooperativity. This information is usually insufficient to provide the basis for mechanistic models. In the case of transport using two Na⁺ ions, an n < 2 indicates that molecules with only one of the two sites occupied are present at low saturation. Here, we propose a new way of analyzing Na⁺-binding data for the case of two Na⁺ ions that, by taking into account binding to individual sites, provides far more information than can be obtained by using the Hill equation with a non-integer coefficient: it yields pairs of possible values for the Na⁺ affinities of the individual sites that can only vary within narrowly bounded ranges. To illustrate the advantages of the method, we present experimental scintillation proximity assay (SPA) data on binding of Na⁺ to the Na⁺/I⁻ symporter (NIS). SPA is a method widely used to study the binding of Na⁺ to Na⁺-driven transporters. NIS is the key plasma membrane protein that mediates active I⁻ transport in the thyroid gland, the first step in the biosynthesis of the thyroid hormones, of which iodine is an essential constituent. NIS activity is electrogenic, with a 2:1 Na⁺/I⁻ transport stoichiometry. The formalism proposed here is general and can be used to analyze data on other proteins with two binding sites for the same substrate.