Sodium Aspartate Research Articles

Fractional recording and component analysis of primate LERG: Separation of photoreceptor and other retinal potentials

The local electroretinogram, as obtained with a single micro electrode in the rhesus monkey fovea is known to be the sum of a number of components. This paper shows how the receptor component may be isolated from the LERG by using a bipolar micro electrode that records the potential across the outer segments of the foveal cones. A component analysis of the single electrode LERG, performed at a number of electrode depths in the retina, yields four components: a receptor-, b-, dc- and slow component. The latter has not been reported previously in the monkey and it is proposed that this is the slow PIII of the primate retina. The amplitude-depth profiles of the four components show why the receptor component may be isolated by fractional recording. The recording area of a bipolar electrode is 10 times smaller than that of a monopolar electrode. Contribution of rods to the responses is often present in the dark adapted foveal LERG but is absent in fractional records from the central fovea. The infusion of sodium aspartate in the eye abolishes the b-component, but does not affect the slow component. Aspartate produces, however, such further complexities that the method seems not suitable for isolation of the receptor potential in the intact primate eye.

Lead-induced experimental lesions of the testis and their treatment.

Thirty-six healthy albino rats were divided into three groups and the histologic structure of the testes was examined after treatment: group 'C'--10 healthy control rats were untreated; group 'Pb'--eight rats received intraperitoneal injections of 8.5 mg per kg body weight lead acetate for 154 days (15 doses in all); group 'PbA'--eight rats intoxicated with lead, like group 'Pb' but which, during the last 21 days before killing, were treated with 5 ml per kg body weight per day of a 2% injectable solution of sodium aspartate. As compared with the controls, group 'Pb' showed alteration of the histopathologic pattern and reduction in the size of seminiferous tubules, basal membrane separation and a decrease in the cell content of the germinal layer, lesions of the spermatocytes and spermatids, and slight oedematous dissociation in the interstice. In 75% of the group 'PbA' animals, normal aspects similar to those of group 'C' were found.

Stereospecificity of sodium borohydride reduction of Schiff bases at the active site of aspartate aminotransferase.

Sodium boro[3H]hydride treatment of holoaspartate aminotransferase results in the reduction of the Schiff's base formed between pyridoxal phosphate and Lys 258. Treatment of the reduced enzyme with papain followed by acid hydrolysis liberates epsilon-N-[3H]pyridoxyl lysine which is degraded to [3H]pyridoxamine diHCl and stereochemically analyzed with apoaspartate aminotransferase. Sodium boro[3H]hydride treatment of active site carbamylated aspartate aminotransferase reconstituted with pyridoxyl phosphate and sodium aspartate results in the trapping of an enzyme x substrate complex through the reduction of the Schiff's base formed between pyridoxal phosphate and aspartate. Active site bound N-[3H]pyridoxyl aspartate is liberated by treatment with papain and degraded to [3H]pyridoxamine diHCl for stereochemical analysis. Borohydride reduction of the holoenzyme occurs from the re face of the pyridoxal phosphate Lys 258 Schiff's base. Similarly, reduction of active site carbamylated enzyme x substrate complex occurs from the re face of the pyridoxal phosphate-aspartate Schiff's base. These results indicate that when active site carbamylated enzyme binds substrate to pyridoxal phosphate it does so stereospecifically and without changing the face of the Schiff base that is available for reduction as compared to native enzyme.

The sodium effect of Bacillus subtilis growth on aspartate.

aspH mutants of Bacillus subtilis have a constitutive aspartase activity and grow well on aspartate as sole carbon source. aspH aspT mutants, which are deficient in high affinity aspartate transport as a result of the aspT mutation, grow as well as aspH mutants in medium containing high concentrations of aspartate and Na+. This Na+ effect is not due to an enhancement of aspartate transport but is the result of increased cellular metabolism. The ability to grow rapidly in sodium aspartate is induced by prior growth in the presence of Na+. In potassium aspartate, the addition of arginine, citrulline, ornithine, delta 1-pyrroline-5-carboxylase or proline instead of Na+ also allows rapid growth; but in a mutant deficient in ornithine--oxo-acid aminotransferase, only pyrroline-carboxylate or proline can replace Na+. The amino acid pool of cells growing slowly in potassium aspartate contains proline at a low concentration which increases upon addition of proline (but not Na+) to the medium. Thus, Na+ addition does not increase the synthesis of proline, but proline or pyrroline-carboxylate acts similarly to Na+ either in preventing some inhibitory effect (by aspartate or the accumulating NH4+) or in overcoming some deficiency (e.g. in further proline metabolism.

Acetylcholine-induced current in perfused rat myoballs.

Spherical "myoballs" were grown under tissue culture conditions from striated muscle of neonatal rat thighs. The myoballs were examined electrophysiologically with a suction pipette which was used to pass current and perfuse internally. A microelectrode was used to record membrane potential. Experiments were performed with approximately symmetrical (intracellular and extracellular) sodium aspartate solutions. The resting potential, acetylcholine (ACh) reversal potential, and sodium channel reversal potential were all approximately 0 mV. ACh-induced currents were examined by use of both voltage jumps and voltage ramps in the presence of iontophoretically applied agonist. The voltage-jump relaxations had a single exponential time-course. The time constant, tau, was exponentially related to membrane potential, increasing e-fold for 81 mV hyperpolarization. The equilibrium current-voltage relationship was also approximately exponential, from -120 to +81 mV, increasing e-fold for 104 mV hyperpolarization. The data are consistent with a first-order gating process in which the channel opening rate constant is slightly voltage dependent. The instantaneous current-voltage relationship was sublinear in the hyperpolarizing direction. Several models are discussed which can account for the nonlinearity. Evidence is presented that the "selectivity filter" for the ACh channel is located near the intracellular membrane surface.

Placental transfer of aspartate and its metabolites in the primate

The placental transfer of aspartate was tested in pregnant monkeys infused maternally with sodium aspartate. In five animals infused at 100 mg/kg/hr, maternal plasma aspartate levels increased from 0.36 ± 0.19 to 80.2 ± 11.5 μmole/dl (mean ± SD). However, fetal plasma aspartate levels increased only slightly from 0.42 ± 0.31 to 0.98 ± 0.24 μmole/dl ( p = 0.02). Erythrocyte aspartate levels were unchanged in both fetal and maternal circulation. In two animals infused at 200 mg/kg/hr, maternal plasma aspartate levels increased from 0.28 and 0.31 μmole/dl to values of 141 and 237 μmole/dl, respectively. This increase produced a significant ( p = 0.001) increase in fetal plasma aspartate levels from 0.53 and 0.67 to 3.3 and 4.5 μmole/dl, respectively. Maternal plasma aspartate levels in two animals infused at 400 mg/kg/hr increased from 0.5 and 0.7 μmole/dl to 400 and 750 μmole/dl, respectively, at the end of the infusion. Fetal plasma aspartate levels increased from 0.21 and 0.25 μmole/dl to 60 and 92 μmole/dl, respectively. Maternal aspartate infusion at each level increased maternal, but not fetal, plasma taurine levels. The increase in maternal taurine levels was not in proportion to the dose of aspartate infused. Aspartate metabolites, glucose, and lactate were readily transferred across the placenta. The data indicate that aspartate, like glutamate but unlike most amino acids, is not concentrated toward the fetal circulation in the pregnant primate, and suggest that a barrier to aspartate transfer exists unless maternal plasma levels are grossly elevated.

The increase in sensitivity following light illumination in frog photoreceptors

Dark-adaptation of photoreceptors was studied by recording fast PIII responses of the isolated bull frog retina superfused with Conway's solution containing 5 mM sodium aspartate. When a dark-adapted retina is illuminated by an adapting light, the amplitude of the response decreases. Initially on turning off this light, the amplitude increases over that of the dark-adapted response. This phenomenon, “hypersensitivity” is thought to be due to an increase in sensitivity of red rods. The hypersensitivity occurs following several minutes illumination with relatively weak light if [Ca 2+ out is low. Intense light and higher concentration of Ca 2+ inhibit the hypersensitivity. Possible mechanisms for the hypersensitivity are discussed.

Combined effect of veratridine and sodium aspartate on the rabbit retinas in vitro.

Combined effect of veratridine and sodium aspartate on rabbit retinas in vitro was studied. Observations suggested that mode of action of veratridine on the PII and PIII components of ERGs was modified by the presence of sodium aspartate in perfusate.

Intracellular recordings from gecko photoreceptors during light and dark adaptation.

Intracellular recordings were obtained from rods in the Gekko gekko retina and the adaptation characteristics of their responses studied during light and dark adaptation. Steady background illumination induced graded and sustained hyperpolarizing potentials and compressed the incremental voltage range of the receptor. Steady backgrounds also shifted the receptor's voltage-intensity curve along the intensity axis, and bright backgrounds lowered the saturation potential of the receptor. Increment thresholds of single receptors followed Weber's law over a range of about 3.5 log units and then saturated. Most of the receptor sensitivity change in light derived from the shift of the voltage-intensity curve, only little from the voltage compression. Treatment of the eyecup with sodium aspartate at concentrations sufficient to eliminate the beta-wave of the electroretinogram (ERG) abolished initial transients in the receptor response, possibly indicating the removal of horizontal cell feedback. Aspartate treatment, however, did not significantly alter the adaptation characteristics of receptor responses, indicating that they derive from processes intrinsic to the receptors. Dark adaptation after a strongly adapting stimulus was similarly associated with temporary elevation of membrane potential, initial lowering of the saturation potential, and shift of the voltage-intensity curve. Under all conditions of adaptation studied, small amplitude responses were linear with light intensity. Further, there was no unique relation between sensitivity and membrane potential suggesting that receptor sensitivity is controlled at least in part by a step of visual transduction preceding the generation of membrane voltage change.

Light adaptation of the receptors: Increment threshold functions for the frog's rods and cones

Using sodium aspartate Ringer's we recorded gross potentials from the receptors of the frog's isolated retina. With appropriate selection of stimulating conditions, increment threshold functions for the 502 rods and the 580 cones were measured using two test flash durations. All functions indicate a decrease in receptor sensitivity with increases in background light intensity. However, the shape of these functions is dependent upon receptor type and exposure duration. The differences among these functions are discussed. In addition, it is argued that the rods' greater sensitivity to steady background lights is the major cause of their insensitivity, relative to the cones, in the presence of moderate background lights.

Response of primate cones to sinusoidally flickering homochromatic stimuli.

1. The response of the primate cone photoreceptors to sinusoidally flickering stimuli has been obtained by monitoring the late receptor potential (LRP). 2. By comparing the response characteristics of the foveal local electroretinogram (LERG) before and after the intraocular infusion of sodium aspartate, it was found that the b-wave in the foveal LERG does not affect the monitoring of the LRP to steady-state flicker. 3. Functions describing the supra-threshold frequency response characteristics of the photoreceptors were obtained. 4. Linearity was found to hold for low amplitude responses, and temporal modulation transfer functions (MTFs) were obtained for the photoreceptors at various adaptation levels. 5. The cone photoreceptors were found to act approximately as passive low pass filters compounded with some low frequency attenuation. 6. The high frequency response of the photoreceptors at various adaptation levels tends toward a common high frequency asymptote, much like human psychophysical findings, and can be described by a diffusion model. 7. Non-linearities (convexity-upwards) suggest modest positive feedback at the level of the photoreceptors. 8. Mechanisms limiting the magnitude of the receptor response at low frequencies have little effect on the phase lag of the response.

Botulinum Intoxication of Isolated Retinas

Isolated perfused rabbit retinas were subjected to molar concentrations of botulinum toxin type A in the vicinity of 10^––9. For flash light responses, botulinum intoxication produced selective enlargement of the receptor potential (P III) of the electroretinogram. The effects of botulinum intoxication were not seen when the receptor signal was isolated by cold-block or sodium aspartate which depolarizes the inner layers. Current evidence indicates that botulinum toxicity affects the presynaptic cholinergic endings early in the visual pathway.

Intraretinal isolation of PIII subcomponents in the isolated rabbit retina after treatment with sodium aspartate

By treating the isolated rabbit retina with a plasma-Tyrode mixture containing sodium aspartate the cornea-negative component PIII was intraretinally subdivided into three subcomponents. These subcomponents have been named; (1) an aspartate-insensitive distal PIII, (2) an aspartate-sensitive PIII and (3) an aspartate-insensitive slow PIII. The aspartate-insensitive distal PIII was recorded in the receptor layer after application of aspartate; it is assumed to be the receptor potential. The aspartate-sensitive PIII is eliminated by aspartate, it probably corresponds to the proximal PIII of the frog. The aspartate-insensitive slow PIII can be found during the influence of aspartate proximal to the receptor potential and extends to a depth where the cornea-positive component PII is present if the isolated rabbit retina is perfused with a control solution.

Recovery of cone receptor activity in the frog's isolated retina

Using sodium aspartate Ringer's we recorded gross potentials from the receptors of the frog's isolated retina. With appropriate selection of stimulating conditions, dark adaptation of the 580 cones was studied. After intense lights that bleach substantial cone pigment and raise cone threshold over 2 log units, the cones recover nearly all their sensitivity, within 0.16 log unit. This is in marked contrast to the 502 rods (see following paper) and is consistent with Goldstein's ERP evidence that indicates 580 cone pigment regeneration in the isolated retina.

Photoreception by a cephalopod retina in vitro

The retina of Sepiola atlantica equilibrates in artificial plasma with 60% of its total water accessible to [ 14C]inulin. Subsequent calculations yield intracellular concentrations of 245 m m-sodium and 236 m m-potassium, compared with corresponding external concentrations of 460 and 20 m m. These concentrations yield Nernst potentials of +17 and −63 mV for sodium and potassium respectively and the former is consistent with voltage clamp data from Limulus and barnacle photoreceptors. The Sepiola electroretinogram is immune to tetrodotoxin or sodium aspartate interference, suggesting a purely photoreceptor origin. The isolated retina produces ERG responses to ultra-short (6 μsec) stimuli, comprising a latency phase, a rapid establishment of potential, and a slower decline to the resting level. The established potential is consistent with a flow of positive current into the outer segment region of the photoreceptor cells. The response is quickly supressed by 10 −4 m-ouabain, but is protected when exposure to ouabain occurs in a sodium-free medium, suggesting that electrogenic systems are not primarily involved. Reduction in external sodium concentrations, or increase in potassium, both reduce the response, consistent with a sodium conductance increase driving the ERG. Substitution of propionate for external chloride changes the response, possibly by a positive effect. Absence of external calcium markedly retards the decline phase of the ERG, implicating calcium in closing the notional sodium channels. At low temperatures the whole ERG sequence is retarded.

Dark adaptation of the frog's rods

Using sodium aspartate Ringer's and isolated frog retinas we have recorded gross potentials from the frog's 502 (rhodopsin) rods. Substantial dark adaptation is seen at the receptor in the isolated retina in which little or no rod pigment regeneration occurs. Lights that bleach only a fraction of a per cent of rod pigment substantially decrease rod sensitivity, but sensitivity quickly returns to the prebleach level. With lights that bleach measurable amounts of pigment, the rods recover their sensitivity more slowly and show a permanent loss in sensitivity.

Adaptation in skate photoreceptors.

Receptor potentials were recorded extracellularly from the all-rod retina of the skate after the application of sodium aspartate. This agent suppresses the responses of proximal elements, but leaves relatively unaffected the electrical activity of the photoreceptors (a-wave) and pigment epithelium (c-wave). Since the latter develops too slowly to interfere with the receptor response, it was possible to isolate receptor potentials and to compare their behavior in light and dark adaptation with earlier observations on the S-potential, b-wave, and ganglion cell discharge. The results show that the photoreceptors display the full complement of adaptational changes exhibited by cells proximal to the receptors. Thus, it appears that visual adaptation in the skate is governed primarily by the photoreceptors themselves. Of particular interest was the recovery of sensitivity in the presence of background fields that initially saturate the receptor potential. Analysis of this recovery phase indicates that a gain-control mechanism operates within the receptors, at a distal stage of the visual process.

The isolated receptor potential of the frog isolated retina: Action spectra before and after extensive bleaching

We have used sodium aspartate Ringer's to enable us to record gross potentials from the receptors of the frog's isolated retina. After bleaching 92 per cent of the rhodopsin, at least two receptors recover their sensitivity, although no rhodopsin is regenerated. Selective chromatic adaptation was used to demonstrate that the receptors involved have λ max around 500 and 580 nm. One receptor must be the 580 cone. The other receptor has an action spectrum fitted by a 502 nomogram pigment and must be the red rod or accessory cone. The implications of these recoveries in sensitivity are discussed.

The effect of aspartate on the ERG of the isolated rabbit retina.

In 1969 Sillman, Ito and Tornita reported on the effect of sodium aspartate on the ERG of the isolated frog retina. Besides abolition of the b-wave (which was described earlier by Furukawa and Hanawa in the toad, 1955) the authors succeeded in separating distal and proximal PIII (Murakami and Kaneko, 1966) by application of aspartate. By the suppression of the proximal PIII the receptor potential (distal PIII) could be isolated.

Studies on the mass receptor potential of the isolated frog retina: II. On the basis of the ionic mechanism

The receptor potential of the excised frog retina was isolated by treatment of the tissue with Ringer solutions containing sodium aspartate, and was recorded by means of a pair of external electrodes on the opposite sides of the retina. Except at very low concentrations, the amplitude of the receptor potential varied in direct linear proportion to the logarithm of the external sodium concentration, and in inverse linear proportion to the logarithm of the external potassium concentration. The receptor potential could be generated by light even after the metabolic mechanism has been suppressed by ouabain, providing that a sodium concentration gradient was maintained. It was concluded that the primary action of light in generating the receptor potential is one of decreasing the permeability of the photoreceptor membrane to sodium. The metabolic pump is important only in maintaining the normal sodium concentration gradient. A model in the outer segment was proposed to explain the ionic mechanism in terms of the present and previous studies.