Time Course Of Changes In Concentration Research Articles

Stability, Disposition, and Penetration of Catalytic Antioxidants Mn-Porphyrin and Mn-Salen and of Methylprednisolone in Spinal Cord Injury

This study measured the time courses of concentration changes following administration of the catalytic antioxidants Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) and Mn (III) 3-methoxy N, N' bis (salicyclidene) ethylenediamine chloride (EUK-134) in blood and cerebrospinal fluid (CSF) of rats with a spinal cord injury (SCI) and sham controls. Parallel measurements were made for methylprednisolone, the only drug presently used clinically for treating SCI. The time courses kinetically characterized the agents in their stability, disposition, and ability to penetrate the blood-spinal cord barrier (BSB). In both the SCI and control groups, MnTBAP was stable in CSF and in blood across the collection periods (10 h and 24 h, respectively) following administration. In the blood, [EUK-134] and [methylprednisolone] rapidly declined to near basal concentrations at 4 h and 2 h, respectively, post-administration. Therefore the order of stability in CSF and blood was MnTBAP >> EUK-134 > methylprednisolone. The maximum CSF/blood concentration ratios for EUK-134, methylprednisolone and MnTBAP post-administration were: 32 ± 3.1%, 19.2 ± 6.4%, and 4.42 ± 0.73% in the injured rats, and 22 ± 6.5%, 17.8 ± 2.9%, and 1.0 ± 0.5% in the sham control animals. This suggests an order of BSB penetration of EUK-134 > methylprednisolone >> MnTBAP. Despite much lower penetration by MnTBAP compared with EUK-134 and methylprednisolone, a lower dose of MnTBAP because of its stability provided a higher concentration in CSF than did the other agents given at higher doses. This finding supports further exploration of MnTBAP as a potential treatment for SCI.

Newly developed Mg2+-selective fluorescent probe enables visualization of Mg2+ dynamics in mitochondria.

Mg2+ plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg2+ regulation and the Mg2+ concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg2+ in mitochondria in intact cells. Here, we have developed a novel Mg2+–selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg2+ concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg2+, KMG-104, enabled us to compare the dynamics of Mg2+ in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)–induced Mg2+ mobilization from mitochondria to the cytosol was visualized. Although a FCCP–induced decrease in the Mg2+ concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg2+ and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP+). A gradual decrease in the Mg2+ concentration in mitochondria was observed in response to MPP+ in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg2+ dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).

The dynamics of cysteine, glutathione and their disulphides in astrocyte culture medium

Glutathione (GSH) plays an important neuroprotective role, and its synthesis depends on the amount of available cysteine (CSH) in the cells. Various kinds of evidence suggest that astrocytes can provide CSH or GSH to neurons, but the delivery mechanism of the thiol-compounds has not been elucidated. In this study, the dynamics of CSH, GSH and their disulphides in astrocyte culture medium were investigated by following the time-course of concentration changes and by computer simulation and curve fitting to experimental data using a mathematical model. The model consists of seven reactions and three transports, which are grouped into four categories: autoxidation of thiols into disulphides, thiol-disulphide exchange and reactions of thiols with medium components, as well as the cellular influx and efflux of thiols and disulphides. The obtained results are interpreted that cystine (CSSC) after entering astrocyte is reduced to CSH, most of which is released to medium and autoxidized to CSSC. The efflux of GSH was estimated to be considerably slower than that of CSH, and most of the excreted GSH is converted to cysteine-glutathione disulphide principally through the thiol-disulphide exchange. The results seem to indicate that astrocytes provide neurons mainly with CSH, rather than GSH, as the antioxidant material for neuroprotection.

Estimation of the postmortem interval by means of 1H MRS of decomposing brain tissue: influence of ambient temperature

Standard methods for the estimation of the postmortem interval (PMI, time since death), based on the cooling of the corpse, are limited to about 48 h after death. As an alternative, noninvasive postmortem observation of alterations of brain metabolites by means of (1)H MRS has been suggested for an estimation of the PMI at room temperature, so far without including the effect of other ambient temperatures. In order to study the temperature effect, localized (1)H MRS was used to follow brain decomposition in a sheep brain model at four different temperatures between 4 and 26°C with repeated measurements up to 2100 h postmortem. The simultaneous determination of 25 different biochemical compounds at each measurement allowed the time courses of concentration changes to be followed. A sudden and almost simultaneous change of the concentrations of seven compounds was observed after a time span that decreased exponentially from 700 h at 4°C to 30 h at 26°C ambient temperature. As this represents, most probably, the onset of highly variable bacterial decomposition, and thus defines the upper limit for a reliable PMI estimation, data were analyzed only up to this start of bacterial decomposition. As 13 compounds showed unequivocal, reproducible concentration changes during this period while eight showed a linear increase with a slope that was unambiguously related to ambient temperature. Therefore, a single analytical function with PMI and temperature as variables can describe the time courses of metabolite concentrations. Using the inverse of this function, metabolite concentrations determined from a single MR spectrum can be used, together with known ambient temperatures, to calculate the PMI of a corpse. It is concluded that the effect of ambient temperature can be reliably included in the PMI determination by (1)H MRS.

Visualizing localized dynamic changes during epileptic seizure onset in vivo with diffuse optical tomography

Diffuse optical tomography (DOT) is a promising functional imaging modality due to its ability to provide quantitative and dynamic tomographic imaging of brain functions. This pilot study was conducted to demonstrate that DOT can be used to visualize the changes in local hemodynamics during seizures. The focal seizure was induced by microinjection of 10 microl of 1.9 mM GABAA antagonist bicuculline methiodide (BMI) into the left parietal neocortex of male Harlen Sprague-Dawley rats, which was imaged by a multispectral continuous-wave DOT system. Functional images were obtained by our finite element based image reconstruction algorithm. A series of dynamic 2D images were obtained to delineate the time course of concentration changes of oxyhaemoglobin, deoxyhaemoglobin, and total hemoglobin in the rat brain during seizure onset. The BMI induced epileptic foci were localized and observed over time from the images obtained. Our results suggest that diffuse optical tomography may be a promising modality for epilepsy imaging due to its ability to localize epileptic foci as well as its potential to map the functional activity in the area of human cerebral cortex in planning of epilepsy surgery.

Equilibrium of Nucleotides in the Dogfish Brain

In the past, the results of experiments on the time course of concentration changes of adenylates, phosphocreatine, and free creatine in muscle appeared compatible with an equilibrium hypothesis involving only the Lohmann and the myokinase reactions. Other reports, however, denied the applicability of the equilibrium hypothesis to the same tissue. The controversy may have been due to the high probability of experimental errors since time sampling was performed at second intervals. We presently test the hypothesis in the living brain of the small-spotted dogfish shark (Scyliorhinus canicula), an animal-model allowing for timing of sampling at hourly intervals. According to our earlier work, the dogfish shark can easily be resuscitated 8.2 h on average after being brought into the state of "suspended animation" at 0 degree C body temperature and exposed, out of water, to an atmosphere of nitrogen gas. To obtain a complete mathematical description of the time course of concentration changes of brain adenylates and phosphocreatine, we devised a kinetic model based on principles of classical multicompartmental analysis and biochemical kinetics. Model testing of the equilibrium hypothesis resulted in very good agreement between the hypothesis and our experimental data. Time-course modeling, achieved by simultaneously fitting the time series of our data by the set of four equations constituting our model resulted in an excellent agreement between data points and the computed curves. Finally, modeling of the depletion profiles of brain energy status concerning three of its descriptors (energy charge, total adenylate, and primary energy stores expressed in high-energy phosphate equivalents) allowed for a correlation to be established between energy status and the "revival time," a valuable physiological descriptor of tolerance.

Ion Fluxes in Giant Excised Cardiac Membrane Patches Detected and Quantified with Ion-selective Microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2–3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10–15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion–coupled transporters in giant patches.

Cytochemical localization of guanylate cyclase in photoreceptor cells of the mouse.

Light-stimulated excitation causes a decrease of the cGMP concentration in vertebrate photoreceptor cells. The cGMP content is restored by the catalytic action of a guanylate cyclase (EC 4.6.1.2). The spatial distribution of guanylate cyclase was determined cytochemically in rod visual cells of the mouse. In retinal tissue of the mouse guanylate cyclase was found throughout the photoreceptor cells, in the outer and the inner segments, and was especially prominent in the cilia and in elongations of cilia extending into the outer segments. A reaction product of adenylate cyclase (EC 4.6.1.1) could not be demonstrated in vertebrate rod outer segments. The relatively high amount of guanylate cyclase in the inner segments and the cilia may contribute-at least in part-to the actual concentration and the time course of concentration changes of the cGMP concentration in rod outer segments.

Action potential duration modulates calcium influx, Na(+)-Ca2+ exchange, and intracellular calcium release in rat ventricular myocytes.

The experimental work summarized in this paper and described in more detail in our previous publication demonstrates a very important functional role for Na(+)-Ca2+ exchange in intracellular Ca2+ homeostasis in ventricular myocytes from rat hearts. Ca2+ homeostasis in mammalian cardiac myocytes can be considered to be the result of four interactive processes: (i) Ca2+ influx through L-type Ca2+ channels, (ii) Ca2+ release from the SR and its subsequent re-uptake, (iii) intracellular Ca/+ buffering, and (iv) Ca2+ extrusion across the sarcolemma. Our results demonstrate a number of interesting features of these processes. (1) When the action potential voltage-clamp technique is used to identify the size and time-course of Ca2+ fluxes during the action potential, both the peak current and the associated influx of Ca2+ are relatively large as was previously demonstrated by Isenberg and his colleagues. (2) Nevertheless, this source of Ca2+ is unable, by itself, to produce a significant twitch, which is consistent with previous data from rat ventricle. (3) This Ca2+ influx, however, does represent the trigger for SR Ca2+ release. (4) The Na(+)-Ca2+ exchanger on the SR is able, on average, to extrude all the Ca2+ which enters through L-type Ca2+ channels, although it provides relatively little Ca2+, i.e., during the course of the normal action potential there is no significant reverse Na(+)-Ca2+ exchange activity, at least under our experimental conditions. Our results also suggest that although the L-type Ca2+ current cannot by itself trigger and control contraction its amplitude, frequency, and time-course can alter the rate and the extent of Ca2+ release from the SR. Recently, detailed mathematical formulations and a direct demonstration of some of these phenomena have been published. Stern and Stern and Lakatta predicted more than three years ago that the concentration and the time-course of change in concentration of Ca2+ very near the release sites of the SR may be critical determinants of the overall release process. Within the past year Wier and his colleagues and also Lederer et al. have combined electrophysiological measurements with recordings of localized intracellular Ca2+ (made using a confocal microscope) and have shown that rapid, and relatively large, but very localized changes in intracellular Ca2+ due to Ca2+ influx through L-type Ca2+ channels are responsible for triggering, and to some extent, controlling the release of Ca2+ from the SR. However, it has also been shown that this release depends importantly on the loading or priming state of the SR. Perhaps not surprisingly, the massive release of Ca2+ from the SR can, itself, alter the pattern of subsequent SR release events (cf. Ref. 46) and the time-course of Ca2+ influx through the L-type Ca2+ channels. Thus, although our relatively crude measurements have clearly demonstrated the relationship between L-type Ca2+ channel activity and Na+-Ca2+ exchanger function during a normal cardiac action potential in rat ventricle, they fall far short of any delineation of the functional roles of either of these processes in overall Ca2+ homeostasis. This additional information can, in principle, be obtained from studies in which cellular microanatomy can be visualized dynamically in conjunction with localized changes in intracellular Ca2+ as well as Ca2+ of L-type Ca2+ channels, SR release, and cell shortening.

Regulation of rCBF by diffusible signals: An analysis of constraints on diffusion and elimination

AbstractLocal changes in cerebral hemodynamics are observed within a few hundred milliseconds of changes in neural activity. If hemodynamic responses are mediated by passive diffusion of a spatial signal (from the site of neural activity to the microvessels) then the dynamics of the response suggest a lower limit on the signal's apparent diffusion and elimination. The aim of this work was to estimate these limits and narrow the field of possible candidate substances.A simple biophysical simulation was used to examine how the time course of concentration changes in a spatial signal, at the site of action (microvessels), depends on key diffusion parameters (source geometry, apparent diffusion and elimination half‐life). The simulations suggested (1) that the rise in signal concentration is mostly a function of source geometry and diffusion. Conversely falls in concentration depend on elimination and (2) even when sources are very sparsely distributed Nitric Oxide would have a sufficiently fast diffusion and elimination to signal the early components of activity‐dependent hemodynamic response by passive diffusion. © 1995 Wiley‐Liss, Inc.

Influence of metalaxyl on abscisic acid levels in soybean hypocotyls infected with Phytophthora sojae

Previous studies have demonstrated that susceptible soybean plants treated with the acylalanine fungicide, metalaxyl, and inoculated with a compatible race of Phytophthora sojae develop lesions phenotypically similar to those in an incompatible interaction. The present study explores this similarity further by following the time-course of changes in concentrations of glyceollin and abscisic acid in inoculated metalaxyl-treated and untreated plants. Concentrations of metalaxyl were determined by an enzyme-linked immunosorbent assay to be 0·1-0·2 μg g -1 fresh weight in hypocotyl tissues of soybean seedlings treated by root uptake for 16 h with 1 μg ml -1 metalaxyl. These tissue concentrations are sufficient to cause 20-50 percent growth inhibition in vitro of P. sojae. Following inoculation with a compatible race of P. sojae, restricted lesions containing high levels of glyceollin developed similar to those in an incompatible interaction. During the development of the lesion metalaxyl concentrations within the lesion increased 10-fold in parallel with but preceding by approx. 3 h the time-course of glyceollin accumulation. Abscisic acid levels in the lesions and the tissues immediately surrounding the lesions were measured using an enzyme-linked immunosorbent assay. Abscisic acid concentrations in metalaxyl-treated plants declined rapidly following inoculation with a compatible race of P. sojae. This was similar to the rapid decline in abscisic acid concentration that occurs in untreated plants inoculated with an incompatible race of P. sojae. No comparable changes occurred in treated or untreated plants inoculated with a metalaxyl-tolerant mutant of a compatible race of P. sojae. Thus, the study provides additional evidence that the resistance phenotype induced by metalaxyl treatment duplicates in detail the resistance response that follows recognition of an incompatible race of P. sojae.