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PubMed1984

Ultrastructural localization of acetylcholinesterase in substantia nigra: a comparison between rat and guinea pig.

The distribution and ultrastructural localization of acetylcholinesterase (AChE) was examined in the substantia nigra of rat and guinea pig. Although the pars compacta, in both species, is clearly defined when stained with thionin, there is an apparent discrepancy in the distribution of AChE at the light microscope level. In the rat substantia nigra the enzyme appears to be concentrated mainly in the pars compacta, whereas in the guinea pig the AChE seems homogeneous throughout the entire substantia nigra. Ultrastructural studies, however, reveal a close correspondence in the morphology of rat and guinea pig substantia nigra. The subcellular localization of AChE is also similar. The between-species discrepancy seen with the light microscope was attributed to relative differences in level of intensity of staining for AChE. In both rat and guinea pig, the enzyme is localized in two types of neuron and in the extracellular space. The ultrastructural distribution of AChE is discussed with reference to neurochemical studies on its release from nigral neurons.

Acetylcholinesterase↗
PubMed1975

Synaptic changes in frog brain after stimulation with potassium chloride.

In vitro preparations of frog brains, stimulated by application of CKl were fixed by freeze substitution and examined electron microscopically. Control preparations were bathed in a calcium-free physiological solution with Mg added or in salt solution cooled to 5-10-degrees C. The isolated brain remains viable in the physiological solution as indicated by the direct cortical responses which can be led off from the forebrain. Control preparations were characterized by a row of vesicles situated close to the presynaptic membrane and by the absence of a well-developed postsynaptic web. In KCl stimulated preparations there were, in addition to synapses resembling those in the controls, synapses exhibiting fusion of synaptic vesicles with the membrane of the axonal ending, synapses in which the vesicles had retreated from the presynaptic membrane but were attached to it by a narrow stalk and synapses exhibiting a pronounced postsynaptic web. The synaptic gap was of a less uniform width than in the control preparations. The KCl stimulated preparations were furthermore characterized by a paucity of extracellular space and often showed invaginations formed by the presynaptic membrane and the plasma membranes of the postsynaptic or adjacent glial structure.

Animals↗
PubMed1959

Muscle: a three phase system. The partition of divalent ions across the membrane.

The partition of sulfate, Ca(++), and Mg(++) across the membrane of the sartorius muscle has been studied, and the effect of various concentrations of these ions in the Ringer solution on the cellular level of Na(+), K(+), and Cl(-) has been determined. The level of the three divalent ions in toad plasma and muscle in vivo has been assayed. Muscle was found to contain an almost undetectable amount of inorganic sulfate. Increases in the external level of these ions brought about increases in intracellular content, calculated from the found extracellular space as determined with radioiodinated serum albumin or inulin. Less of the cell water is available to sulfate than to Cl(-), and the Mg(++) space is less than the Na(+) space. An amount of muscle water similar to that found for Li(+) and I(-) appears to be available to these divalent ions. Sulfate efflux from the cell was extremely rapid, and it was not found possible to differentiate kinetically between intra- and extracellular material. These results are consistent with the theory of a three phase system, assuming the muscle to consist of an extracellular phase and two intracellular phases. Mg(++) and Ca(++) are adsorbed onto the ordered phase, and increments in cellular content found on raising the external level are assumed to occur in the free intracellular phase.

Calcium↗
PubMedDate not supplied

Brain interstitial fluid and intracellular distribution of phenytoin.

After intravenous (i.v.) administration (10 mg/kg), the biodisposition of phenytoin (PHT) in serum (total and free concentration), cerebrospinal fluid (CSF), brain, and the interstitial fluid (IF) of the normal brain were determined in dogs. A sufficient volume of IF was obtained through a multiperforated polypropylene ball implanted into the left parietotemporal region for 4-5 weeks. PHT brain distribution coefficient values ranged between 1.9 and 3.75, while the ratios of IF to free serum PHT concentrations ranged between 0.19 and 1.04; thus, our data indicate that most of the free unbound PHT which enters the brain parenchyma accumulates in the cellular compartment. Furthermore, at 60 and 90 min the peak CSF and IF concentrations are delayed; thus, for PHT, an apparent diffusion front from the CSF into the extracellular space of the brain seems to occur.

Animals↗
PubMed1988

Drug "diffusion" within the brain.

1. The rates and the ways different materials move from ventricular fluid into and through the caudate nucleus vary rather broadly. 2. The major mechanism of solute flow from ventricular CSF into and through the caudate nucleus appears to be simple diffusion. 3. Among solutes that pass across the BBB from brain to blood at very low rates, those that distribute mainly or exclusively in the extracellular space (e.g., sucrose, EDTA, and sodium) "diffuse" most rapidly into the caudate nucleus. 4. Among solutes which efflux across the BBB at very low rates, the apparent rate of diffusion within the caudate nucleus is the least for those that are readily taken up and accumulated by brain cells (e.g., AIB). 5. Solutes that move moderately to rapidly across the BBB from brain to blood only penetrate distances of several mm or less into the caudate before they are completely cleared from the tissue by the cerebral circulation. 6. In most instances the slopes of the tissue concentration profiles within the caudate nucleus during and after ventriculocisternal perfusion are fairly steep and are complex functions of parameters such as the effective tissue diffusion coefficient (Dt) and the transcapillary efflux rate constant (Ko), each of which are, in turn, dependent on separate sets of variables (e.g., the rate of cellular uptake for Dt and the rate of blood flow for ko). 7. The distribution process and kinetics may be much different in CNS areas adjacent to the subarachnoid space than in the caudate nucleus because of convective flow of subarachnoidal CSF through the perivascular spaces of the penetrating and emerging blood vessels and the arterioles and venules.

Animals↗
PubMed1998

Accessibility of T-tubule vacuoles to extracellular dextran and DNA: mechanism and potential application of vacuolation.

In addition to its function in excitation-contraction coupling, the ability of the T-system of skeletal muscle fibres to undergo reversible vacuolation indicates that it may play a role in volume regulation. The mechanism of reversible vacuolation has been investigated by confocal microscopy using fluorescein dextran to probe the accessibility of T-tubules to the extracellular environment. Vacuolation was induced by loading the fibres with 60-100 nM glycerol for 30 minutes and then returning them to glycerol-free medium. Devacuolation was subsequently induced by the reentry of glycerol. During their formation from T-tubules, the vacuoles filled with fluorescent dextran from the extracellular medium. The inaccessibility of the vacuoles to extracellular ferritin observed in a previous study raised the possibility that the vacuoles may be detached from the surface membrane after their formation. However, it is apparent from the present work that, although the tubules of the treated fibres are constricted, the vacuoles maintain a open connection with the external medium for smaller macromolecules. In the light of these experiments, it is proposed that vacuolation is caused by water moving into T-tubules from the cytoplasm faster than it can exit to the extracellular space during a decrease in fibre volume. Since T-tubules have been implicated in the transfection of skeletal muscle by direct injection, the accessibility of plasmid DNA to T-tubules has also been investigated. DNA penetrated into the vacuoles from the extracellular medium less well than dextran, but many vacuoles containing fluorescent DNA were observed in the superficial layers of vacuolated fibres, and it is suggested that T-tubule vacuolation might be used to improve the efficiency of the transfection of skeletal muscle by direct injection.

Animals↗
PubMed1996

Water compartmentalization and extracellular tortuosity after osmotic changes in cerebellum of Trachemys scripta.

1. Water compartmentalization in the turtle cerebellum subject to media of different osmolalities was quantified by combining extracellular diffusion analysis with wet weight and dry weight measurements. The diffusion analysis also determined the tortuosity of the extracellular space. 2. Isolated cerebella were immersed in normal, oxygenated physiological saline (302 mosmol kg-1), hypotonic saline (238 mosmol kg-1) and a series of hypertonic salines (up to 668 mosmol kg-1). The osmolality was varied by altering the NaCl content. 3. Extracellular volume fraction and tortuosity of the granular layer of the cerebellum were determined from measurements of ionophoretically induced diffusion profiles of tetramethylammonium, using ion-selective microelectrodes. The volume fraction was 0.22 in normal saline, 0.12 in hypotonic medium and 0.60 in the most hypertonic medium. Tortuosity was 1.70 in the normal saline, 1.79 in the hypotonic and 1.50 in the most hypertonic saline. 4. The water content, defined as (wet weight-dry weight)/wet weight, of a typical isolated cerebellum (including granular, Purkinje cell and molecular layers) was 82.9%. It increased to 85.2% in hypotonic saline and decreased to 80.1% in the most hypertonic saline. 5. Measurements of extracellular volume fraction and water content were combined to show that hypotonic solutions caused water to move from the extracellular to the intracellular compartment while hypertonic solutions caused water to move from the intracellular to extracellular compartment, with only a relatively small changes in total water in both cases. 6. These results suggest the use of the isolated turtle cerebellum as a model system for studying light scattering or diffusion-weighted magnetic resonance imaging.

Animals↗
PubMed2001

Inhibition of H2 histamine receptor-mediated cation channel opening by protein kinase C in human promyelocytic cells.

Histamine, through H(2) receptors, triggers a prominent rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in addition to an elevation of cAMP level in HL-60 promyelocytes. Here we show that the histamine-induced [Ca(2+)](i) rise was due to influx of Ca(2+) from the extracellular space, probably through nonselective cation channels, as incubation of the cells with SKF 96365 abolished the histamine-induced [Ca(2+)](i) rise, Na(+) influx, and membrane depolarization. The Ca(2+) influx was specifically inhibited by pretreatment of the cells with PMA or extracellular ATP with 50% inhibitory concentrations of 0.12 +/- 0.03 nM and 185 +/- 17 microM, respectively. Western blot analysis of protein kinase C (PKC) isoforms revealed that PMA (< or =1 nM) and ATP (300 microM) caused selective translocation of PKC-delta to the particulate/membrane fraction. Costimulation of the cells with histamine and SKF 96365 partially reduced histamine-induced granulocytic differentiation, which was evaluated by looking at the extent of fMet-Leu-Phe-induced [Ca(2+)](i) rise and superoxide generation. In conclusion, nonselective cation channels are opened by stimulation of the H(2) receptor, and the channels are at least in part involved in the induction of histamine-mediated differentiation processes. Both effects of histamine were selectively inhibited probably by the delta isoform of PKC in HL-60 cells.

Adenosine Triphosphate↗
PubMed1990

[Electron histochemical detection of intracellular and extracellular cathepsin D in the liver].

Cathepsin D localization was studied in the liver of white rats by ultrastructural cytochemistry. It was shown that the product of reaction was present in lysosomes of hepatocytes, Kupffer's and endothelial cells and in fibroblasts from portal tracts am small granules or their conglomerate of different electron density. The lowest activity of cathepsin D was observed in hepatocytes, the most intensive reaction--in Kupffer cells. The extracellular activity of cathepsin D in vivo was revealed. It means that besides participation in intracellular degradation of different proteins, cathepsin D is secreted to extracellular space by liver cells (hepatocytes, Kupffer cells, fibroblasts) and it may participate in catabolism of intercellular matrix.

Animals↗
PubMed1984

[Changes in total body potassium and ion distribution compartments in general surgery and intensive care patients].

Measurements of total body potassium in a whole body counter revealed significant total body potassium depletion in patients suffering from high output enteric fistulas, short bowel syndrome and Crohn's disease. The clinical importance of this finding is derived from the fact that the degree of potassium depletion was found to be related to mortality, and that the introduction of an infusional regimen for preparation for surgery in Crohn's disease which effected intracellular potassium refilling resulted in a decreased surgical mortality and complication rate. In conclusion specific nutritional support as an essential measure in preparing such patients for elective surgery is particularly recommended. Significant cellular potassium depletion was also found in critically ill patients dying from sepsis. In contrast to the aforementioned diseases with malnutrition, where an increased fecal loss of potassium is the most likely cause of potassium deficiency, a different mechanism for cellular potassium loss is suggested in this condition from additional indicator dilution studies. Rather here the finding of increased distribution volumes for ions physiologically predominantly restricted to the extracellular space (82Br and 24Na) indicates a change of cell membran permeability suggestive of so called sick cell syndrome.

Adult↗
PubMed1994

Cross-talk of the receptors for bradykinin, serotonin, and ATP shown by single cell Ca2+ responses indicating different modes of Ca2+ activation in a neuroblastoma x glioma hybrid cell line.

Modes of Ca2+ activation by bradykinin, serotonin, and ATP and the possible receptor cross-talk were investigated in mouse neuroblastoma x rat glioma hybrid cells (108CC15) by monitoring fura-2 fluorescence in single cells. A transient rise of cytosolic Ca2+ activity was induced by short pulses of the hormones. Brief exposure of cells to ionomycin, which depletes intracellular Ca2+ stores, reduced the size of subsequent responses to bradykinin or ATP, but not to serotonin. Superfusion of the cells with Ca(2+)-free medium abolished the Ca2+ response to serotonin, whereas the responses to bradykinin and to ATP were only slightly reduced. This indicates that ATP, like bradykinin, induces the release of Ca2+ from intracellular stores. Serotonin, in contrast, activates Ca2+ entry from the extracellular space. To investigate whether ATP releases Ca2+ from the same stores as bradykinin, we examined the interaction of the hormones by applying them consecutively. When ATP was applied after bradykinin, the nucleotide did not evoke any response, irrespective of the presence or absence of extracellular Ca2+. The application of ATP before that of bradykinin reduced the size of a following bradykinin-induced Ca2+ response in Ca(2+)-free medium, but not in Ca(2+)-containing medium. This suggests that bradykinin may interact with the ATP-activated mechanism by cross-desensitization. Possibly, bradykinin receptors are coupled to additional Ca2+ stores not accessible to ATP that are refilled by extracellular Ca2+. Cyclic AMP and cyclic GMP apparently do not affect the Ca2+ responses to bradykinin and serotonin, as shown by the lack of influence of preincubation of the cells with forskolin or sodium nitroprusside.

Animals↗
PubMed1982

Changes in extracellular potassium and calcium in rat cerebellar cortex related to local inhibition of the sodium pump.

Extracellular K+, Ca2+, and Na+ ([K+]e, [Ca2+]e, [Na+]e) were recorded with ion selective microelectrodes in the cerebellar cortex of urethane-anesthetized rats. Superfusion of the cerebellum with artificial cerebrospinal fluid containing K-strophanthidin (10(-6)-10(-4) mol/l) or other cardioactive steroids, known to be inhibitors of the sodium/potassium pump, had the following effects: elevation of resting [K+]e, reduction of poststimulus K+-undershoots, decrease of resting [Ca2+]e and [Na+]e. For instance, at 3 X 10(-5) mol/l K-strophanthidin within the superfusion solution (the unknown intracerebellar concentration being certainly much smaller), [K+]e was elevated up to 130% and [Ca2+]e reduced to 70% of their resting values. Iontophoretic K+-pulses were enhanced in amplitude at the same time. Control experiments with iontophoretic TMA application demonstrated that the glycoside effects were not due (or in higher concentrations only partly due) to shrinkage of the extracellular fluid volume. When tetrodotoxin (10(-7) mol/l) or Mn2+ (1-3 mmol/l) were additionally superfused, K-strophanthidin effects were qualitatively similar, though quantitatively smaller. This indicates that part of the effects were indirect via neuronal activity evoked by the blockade of the sodium pump. The experiments show that reduction of sodium pump activity in cerebellar cortex has rapid and serious consequences on the distribution of potassium and calcium in the extracellular space, resulting in an alteration of neuronal circuit excitability.

Animals↗
PubMed1994

Posthysteroscopic hyponatremia.

We report a case of severe hyponatremia following hysteroscopic myomectomy for uterine fibroids. This new technique, which requires large volumes of irrigation solution (1.5% glycine in this case), is being more widely applied as an alternative to open surgery. Thus, nephrologists may expect to encounter such cases as the female counterpart to transurethral resection of prostate-associated hyponatremia. Analysis of the dynamic features of this case, in the context of recent knowledge of glycine metabolism, suggests that the early severe hyponatremia is partly spurious, reflecting an osmotic effect of glycine (which is initially restricted to the extracellular space). As glycine progressively enters cells and is metabolized, the later phase of hyponatremia more accurately reflects true hypo-osmolality.

Adult↗
PubMed2000

Regulation of extracellular adenosine in rat hippocampal slices is temperature dependent: role of adenosine transporters.

While a great deal is known about stimuli that can induce the release of adenosine from brain tissue, relatively little is known about the regulation of the basal extracellular concentration of adenosine that is present in the absence of stimulation. Under normal conditions, enough adenosine is present to tonically activate a significant portion of the high-affinity adenosine A1 receptors. The present experiments demonstrated that the estimated basal concentration of extracellular adenosine in rat hippocampal slices maintained at 21 degrees C (430 nM) is approximately twice that at 32 degrees C (220 nM). The sensitivity of presynaptic modulatory adenosine A1 receptors was not significantly different at 21 degrees C or at 32 degrees C. Slices maintained at 21 degrees C also showed a reduced ability to inactivate extracellular adenosine, which reflects a reduction in adenosine transport across cell membranes. This effect appears to be primarily due to a reduction in the function of the equilibrative, dipyridamole-sensitive (ei) adenosine transporter; the nitrobenzylthioinosine-sensitive equilibrative transporter (es transporter) appears to be relatively less affected by temperature than is the ei transporter. These experiments demonstrate that extracellular concentrations of adenosine in the brain are sensitive to temperature, and suggest that some of the neurological effects of hypothermia might be mediated via increased concentrations of adenosine in the extracellular space.

Adenosine↗
PubMed2002

Overexpression of extracellular-SOD in islets of nonobese diabetic mice and development of diabetes.

Mice of the nonobese diabetic strain develop a progressive insulitis resulting in beta-cell destruction and diabetes. Superoxide radicals are abundantly formed by leukocytes and other mechanisms in inflammatory reactions. We here aimed to determine whether superoxide radicals contribute to the beta cell destruction in the mouse model. Transgenic nonobese diabetic mice secreting extracellular-superoxide dismutase under control of the insulin promoter were generated and the development of glucosuria monitored. The overexpression of extracellular-superoxide dismutase resulted in a 6-fold increase in the total superoxide dismutase activity of the islets. The incidence of diabetes of the transgenic mice was, however, not modified. The results suggest that superoxide radicals secreted to the extracellular space do not contribute to the beta cell destruction in the nonobese diabetic mouse model.

Animals↗
PubMed1978

Transfilter lens induction in avian embryo.

The directive influence of the optic vesicle during lens induction of 2-day chick embryos was studied in vitro. Trunk ectoderm was chosen for the responding tissue. This uncommitted ectoderm formed distinct lentoid bodies when grown together with the optic vesicle. Crystallin synthesis was demonstrated in the lentoids with fluorescein labelled antiserum. By interposing filters of different thicknesses and pore sizes between the interacting tissues, three questions were put forward: (1) Whether the intimate contact between the interactants was essential for induction, (2) how far the inductive influence of the optic vesicle extended, and (3) what was the smallest pore through which the inductive influence could penetrate. The inductive influence reached across a Millipore filter with a thickness of 100 micrometer. It also penetrated a dialyzer membrane, allowing passage of molecules with molecular weights (MW) of less than 12,000 daltons. It was concluded that the directive, inductive signal(s) passing from the optic vesicle, diffused in the extracellular space as far as 100 micrometer and had a molecular weight of less than 12,000 daltons.

Animals↗
PubMed1984

Proteins of the brain extracellular fluid: evidence for release of S-100 protein.

Extracellular protein fractions were obtained (1) by mild, isotonic irrigation of freshly perfused brain tissue; (2) by collection of proteins released into superfusing medium by physiologically viable slices of rat hippocampus; and (3) by sampling the CSF of anesthetized rats. Analysis of the S-100 protein content of these fractions gave values of 2.8, 4.2, and 1.8 micrograms S-100/mg protein, respectively. These values were three- to sixfold higher than the S-100 content of the soluble cytoplasmic protein fractions from the same tissue. This several-fold higher S-100 content of the extracellular protein fractions relative to the intracellular cytoplasmic protein fractions indicates that S-100 is selectively released into the extracellular spaces of the brain. We suggest that the biological function of this CNS protein may involve intercellular transfer.

Animals↗
PubMed1996

Postictal alteration of sodium content and apparent diffusion coefficient in epileptic rat brain induced by kainic acid.

PURPOSE: We studied temporal changes of brain sodium and apparent diffusion coefficient (ADC) in a temporal lobe epilepsy (TLE) rat model using kainic acid (KA). METHODS: In situ three-dimensional 23Na magnetic resonance imaging (MRI) and proton diffusion-weighted imaging (DWI) were used. KA at a dose of 10 mg/kg body weight and 12 adult Sprague Dawley rats weighing 228-318 g (268 +/- 25 g) were used. RESULTS: Twenty-four hours after KA injection, magnetic resonance (MR) visible sodium levels increased in both the pyriform cortex (+90%) and amygdala (+68%) and increased insignificantly in the hippocampus (+18%) and caudate-putamen (12%). The ADC in the pyriform cortex showed a -9% decrease at 5 h postictally, reaching -30% at 24 h, whereas in the amygdala decreases were -8 and -26% respectively. A significant decrease in ADC (-7%) in the hippocampus was also observed 24 h postically. Seven days later, sodium increases persisted, whereas ADC returned to normal level. CONCLUSIONS: The increase in MR visible sodium, associated with the decrease in ADC is consistent with the hypothesis that sequential seizures caused an increase in sodium influx and perturbation of membrane ion homeostasis, which eventually evolved into an irreversible phase of cellular edema, with increased MR visible intracellular sodium and decreased ADC. Return of ADC to near-control level and persistent high sodium level at 7 days may be explained by the increase in extracellular space and tissue necrosis.

Amygdala↗