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Intracellular and extracellular compartments of mammalian central nervous tissue.

1. Isolated rabbit retina was used as a prototype of grey matter to study the partition of water and electrolytes between the intracellular and extracellular phase. Previously published morphologic, chemical, and functional evidence has shown that it can be maintained in vitro in a nearly physiological state.2. Following equilibration with mannitol or inulin, retinas were eluted in isotonic tris acetate at 0 degrees C, and measurements were made of the rates at which K(+), Na(+), Cl(-) and inulin or mannitol diffused from the tissue.3. K(+) was eluted slowly according to a single exponential decay. The Na(+) and Cl(-) elution curves demonstrated two phases which could be dissected into a rapid, multicomponent regression superimposed upon a slow exponential decrease.4. The volume of distribution of the readily elutable Na(+) equalled that of the readily elutable Cl(-) and corresponded closely to the volume of distribution of mannitol and inulin. On the basis of these and previously published data, the interstitial fluid was estimated to constitute 31% (w/w) of rabbit retina and 22% of rabbit brain.5. The composition of the extracellular fluid of retina resembled closely that of the medium in which the tissue had been previously incubated.6. Inulin, mannitol, and NaCl diffused through the less accessible portions of the extracellular space at rates which differed from one another as predicted from the diffusion coefficients of these solutes. Their diffusion through the more accessible portions of the extracellular space was apparently affected by bulk flow of the fluid.7. The intracellular concentrations of K(+), Na(+), and Cl(-) were estimated to be 148, 31, and 17 mM respectively.8. Na(+) and K(+) moved across the cell membranes at almost precisely the same rate under the conditions of the elution.

Animals↗

Immunoelectron microscopic localization of albumin in smooth and striated muscle tissues of rat.

Localization of serum albumin in the striated and smooth muscles of rat was studied by an improved immunocytochemical method. Diaphragm, ventricular myocardium, and smooth muscle of stomach were examined. In all of these tissues, albumin was found in the interstitial space and small subsarcolemmal caveolae and vesicles. In addition, the transverse tubular system of the striated muscle stained positive for albumin. The subsarcolemmal vesicles containing albumin did not show any evidence of fusion with lysosomes. Furthermore, in smooth muscle, most of these vesicles were open to the extracellular space. These results demonstrate that albumin in smooth and striated muscle is confined to the extracellular space suggesting that substances such as fatty acids which are carried by albumin are split from it and taken up at the level of the plasma membrane.

Animals↗

Loss of water from heart muscle cells during aging of rats as measured by X-ray microanalysis.

Age-dependent changes of the intracellular water content (IWC) of the rat myocardium have been measured by X-ray microanalysis of deep-frozen bulk specimens (Zs.-Nagy et al., 1982), using a slow warming up and drying of a very superficial layer of the sample, in order to minimize space charging effects. These results were compared with those calculated from the conventionally measured tissue water contents (TWC) and data from literature of morphologically estimated volume density of the extracellular space (David et al., 1981). The IWC values obtained from these two independent methods are in very good agreement, showing that the etching of the surface during the bulk specimen analysis is sufficiently small, i.e., it does not result in any considerable error in the quantitative X-ray microanalysis. The IWC of heart muscle cells decreases significantly with advancing age (from about 80% by weight at the age of 14 days to 71% by the age of 24 months). This observation is consistent with the membrane hypothesis of aging (Zs.-Nagy, 1978). Comparison of the IWC with TWC of the heart muscle shows that the increase of the volume density of the extracellular space during aging balances the age-dependent loss of myocytes to a certain extent.

Age Factors↗

Relationship between astrocytic processes and "perineuronal nets" in rat neocortex.

"Perineuronal nets" (PNs) ensheath a subtype of inhibitory neurons in the mammalian neocortex. In the light of the proposal that PNs consist of glial processes, we have analyzed the relationship between intracellularly injected glial cells and PNs in the rat neocortex. Glial cells were injected iontophoretically with Lucifer Yellow in lightly fixed tissue slices and PNs were visualized with the lectin from Vicia villosa. Using confocal laser scanning microscopy, glial processes and PNs were identified as distinct structures. Lectin labeling was consistently associated with the extracellular space interposed between LY-labeling was consistently associated with the extracellular space interposed between LY-labeled astrocyte processes and neurons. Of the different types of glial cells injected, only the densely-ramifying protoplasmic astrocytes extended processes which could be traced to contact PNs. These protoplasmic astrocytes also sent out processes to adjacent neurons not ensheathed by PNs, and to capillaries. The present data strongly suggests that PNs do not consist of glial processes but rather support the idea that PNs represent specialized extracellular material interposed between the surface of some inhibitory interneurons and astrocytic processes.

Animals↗

Effects of ischaemia and reperfusion on calcium exchange and mechanical function in isolated rabbit myocardium.

We have studied calcium exchanges and mechanical function in heart muscle during and after a period of ischaemia. The experimental preparation was the isolated but arterially perfused interventricular septum of the rabbit. Uptake of calcium was measured with 47Ca2+ and efflux with 45Ca2+. 51Cr-EDTA was used as a marker of the extracellular space. Ischaemia caused a rapid decline of developed tension followed by a rise in resting tension. Tissue counts of 47Ca2+ decreased due to a reduction in the extracellular space. On reperfusion after ischaemia developed tension partially recovered and resting tension increased further before returning towards control values. A large and prolonged uptake of 47Ca2+ occurred immediately on reperfusion while 45Ca2+ efflux rose transiently. The uptake of calcium was related to the severity and duration of ischaemia and to the degree of mechanical recovery. Calcium accumulation on reperfusion is due to an increased influx which is not related to gross disruption of the cell membrane but more probably to a specific abnormality of ionic channels.

Animals↗

Horseradish peroxidase in axons of the dorsal funiculi of the cat: distribution after an injection of the enzyme into the dorsal column nuclei.

Horseradish peroxidase (HRP) was injected into the left dorsal column nuclei of adult cats. Large dorsal funiculi axons of the C3, C5, C8 and L7 segments were searched for HRP-activity after 12, 24, 36 and 48h using light and electron microscopy. Accumulations of intra-axonal HRP-positive bodies occurred at nodes of Ranvier in the C3-C8 segments at 12, 24 and 36h and in the L7 segments at 24, 36, and 48h. The accumulations of HRP in three spatio-temporally different consecutive patterns, noted earlier at nodes of Ranvier in the peripheral nervous system (PNS) portion of feline alpha motor axons for more than 70h after an intramuscular injection of the enzyme, were not observed in the present material. We suggest that the differences in the modes in which large PNS and CNS axons interact with retrogradely transported HRP are due to differences in the organization of the respective nodal regions. We also emphasize that endocytosis via axon terminals in the CNS normally represents uptake of material from an extracellular space which is controlled and protected by the blood-brain barrier. This is in contrast to endocytosis via axon terminals in a muscle, which represents uptake of material from an extracellular space openly exposed to influx of different substances from the blood stream.

Animals↗

Annexin VII relocalization as a result of dystrophin deficiency.

Annexin VII (synexin) is a member of the annexin family of proteins, which are characterized by Ca(2+)-dependent binding to phospholipids. In normal skeletal muscle annexin VII is located preferentially at the plasma membrane and the t-tubule system [Selbert et al. (1995) J. Cell. Sci. 108, 85-95]. Here we have analyzed the distribution of annexin VII in muscle disorders in which the Ca2+ regulation is affected. A remarkable difference was observed in muscle specimens from patients suffering from Duchenne muscular dystrophy and also in muscle from the MDX mouse where annexin VII was gradually released from the sarcolemmal membrane into the cytosol and into the extracellular space during progression of the disease. Hypercontracted muscle fibers positive in Ca2+ staining were devoid of cytosolic annexin VII. Annexins IV and VI were similarly released into the extracellular space. Whereas normal skeletal muscle showed specifically the 51-kDa annexin VII isoform, in dystrophic muscle different ratios of the 51-kDa and the muscle-atypic 47-kDa isoforms were observed. The potential of annexin VII to serve as a tool with which cellular Ca2+ levels can be studied and different muscular disorders classified is discussed.

Animals↗

Effects of chronic digoxin treatment on cardiac function, electrolytes, and sarcolemmal ATPase in the canine failing heart due to chronic mitral regurgitation.

This study was designed to determine whether digoxin therapy in the canine heart failing because of mitral regurgitation (MR) provides only hemodynamic benefit and accompanying subjective improvement or if it also reverses the changes in intracellular Ca++ and sarcolemmal Na+-K+-ATPase. The dogs were divided into four groups: control, MR of 3 months' duration, MR of 6 months', and digoxin treatment for 3 months after 3 months of MR. Six months of MR produced a marked decrease in the index of myocardial contractility and function associated with a decrease in intracellular Ca++ and Na+, and an increase in intracellular K+, extracellular space, sarcolemmal Na+-K+-ATPase, and Mg++-ATPase. Digoxin treatment tended to return the changes in the index of myocardial contractility and cardiac function, intracellular Ca++, Na+, K+, extracellular space, and sarcolemmal Na+-K+-ATPase of the failing heart toward control levels. Digoxin treatment did not affect Mg++-ATPase. The right ventricle, which did not fail, also did not show any significant changes in the parameters measured. The results showed that digoxin treatment not only improved the index of myocardial contractility and cardiac function of the failing heart but also tended to return the electrolytes and sarcolemmal Na+-K+-ATPase toward control levels.

Animals↗

Modification of the blood-brain barrier: increased concentration and fate of enzymes entering the brain.

The blood-brain barrier of rats was opened reversibly by infusing a hyperosmotic solution of arabinose into the external carotid artery. Permeability was increased maximally in the first 15 min and remained slightly elevated at 1 hr. Osmotic barrier opening significantly increased brain uptake of intravenously injected alpha-mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.2.4) (derived from human placenta) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). By injection of 4 X 10(5) units of alpha-mannosidase into an animal, brain activity rose to about twice the normal control activity of the enzyme. After 30 min, activity of administered enzyme in the extracellular space of the brain was calculated to be 30% of the serum concentration. Biochemical and histological studies with horseradish peroxidase showed that exogenously administered enzyme entered the brain extracellular space immediately after barrier opening and was incorporated within neuronal lysosomal packets during the next 24 hr. Measurable peroxidase activity was found in brain as much as 72 hr after osmotic treatment. The results demonstrate that the blood-brain barrier can be reversibly opened to enzymes, that a glycoprotein enzyme is incorporated into neuronal lysosomes, and that the brain may now be considered a potential target for enzyme replacement therapy in heritable metabolic disorders.

Animals↗

Tubular bodies of human endothelial cells in an extracellular location.

The specimen was surgically obtained from a 37 year old female with a cerebellar tumor. The diagnosis of this tumor is still inconclusive, because light microscopic and ultrastructural examination of the tumor showed features that were compatible with either hemangioblastoma or renal cell carcinoma metastatic to the cerebellum. Tubular bodies have heretofore been described as being restricted to an intracytoplasmic location in endothelial cells. Discharge of tubules from these organelles into the vascular lumen and the presence of tubules in the extracellular space between an endothelial cell and a pericyte were observed ultrastructurally in vessels from this tumor. Although this report dealt with only a single pathological case, these findings would support a reasonable postulate of discharge of tubular bodies or their tubules into the extracellular space.

Adenocarcinoma↗

[No free water in the treatment of "hyperosmolar diabetic coma": treatment control by comparing serum and CSF (author's transl)].

Some serum and CSF concentrations were measured in five patients with severe hyperosmolar coma (mean blood sugar = 58.9 mmol/l; osmolarity = 406 mosmol/l). A gradual decrease of serum osmolarity prevented the development of an osmotic gradient between CSF and extracellular space. Insulin treatment (1-20 IU/h) with a motor infusion pump and infusion of hypertonic solutions decreased serum osmolarity by 2-4 mosmol/l X h. The faster fall of glucose in the extracellular space was compensated by hypertonic saline infusions (up to 365 mosmol/l). All patients survived.

Adult↗

Extracellular formation and uptake of adenosine during skeletal muscle contraction in the rat: role of adenosine transporters.

1. The existence of adenosine transporters in plasma membrane giant vesicles from rat skeletal muscles and in primary skeletal muscle cell cultures was investigated. In addition, the contribution of intracellularly or extracellularly formed adenosine to the overall extracellular adenosine concentration during muscle contraction was determined in primary skeletal muscle cell cultures. 2. In plasma membrane giant vesicles, the carrier-mediated adenosine transport demonstrated saturation kinetics with Km = 177 +/- 36 microM and Vmax = 1.9 +/- 0.2 nmol x ml(-1) x s(-1) (0.7 nmol (mg protein)(-1) x s(-1)). The existence of an adenosine transporter was further evidenced by the inhibition of the carrier-mediated adenosine transport in the presence of NBMPR (nitrobenzylthioinosine; 72% inhibition) or dipyridamol (64% inhibition; P < 0.05). 3. In primary skeletal muscle cells, the rate of extracellular adenosine accumulation was 5-fold greater (P < 0.05) with electrical stimulation than without electrical stimulation. Addition of the adenosine transporter inhibitor NBMPR led to a 57% larger (P < 0.05) rate of extracellular adenosine accumulation in the electro-stimulated muscle cells compared with control cells, demonstrating that adenosine is taken up by the skeletal muscle cells during contractions. 4. Inhibition of ecto-5'-nucleotidase with AOPCP in electro-stimulated cells resulted in a 70% lower (P < 0.05) rate of extracellular adenosine accumulation compared with control cells, indicating that adenosine to a large extent is formed in the extracellular space during contraction. 5. The present study provides evidence for the existence of an NBMPR-sensitive adenosine transporter in rat skeletal muscle. Our data furthermore demonstrate that the increase in extracellular adenosine observed during electro-stimulation of skeletal muscle is due to production of adenosine in the extracellular space of skeletal muscle and that adenosine is taken up rather than released by the skeletal muscle cells during contraction.

Adenosine↗

Localization of neural cell adhesion molecule in denervated muscle to both the plasma membrane and extracellular compartments by immuno-electron microscopy.

The location of neural cell adhesion molecule in mouse skeletal muscle has been investigated using two immuno-electron microscopical techniques. In the first method, 6-micron frozen sections of a normal and a hemi-denervated gluteus muscle were stained by an indirect immunoperoxidase method using a rabbit-derived polyclonal antiserum to neural adhesion molecule as the primary antibody. The stain was visualized with the electron-dense chromogen, diaminobenzidine and the sections fixed, osmicated and processed for electron microscopy whilst still on the slide. Ultrathin sections were cut and viewed in the electron microscope, where the reaction product appeared to be localized in the plasma membrane and on the basal lamina of the muscle fibres. The second method, using a 5-nm immunogold-labelled secondary antibody, confirmed and extended these initial observations. In these experiments, the primary antibody to neural cell adhesion molecule was applied in vivo to hemi-denervated mouse gluteus muscles. The muscles were dissected out 24 h later, divided into denervated and innervated halves and then into junctional and extrajunctional regions. Together with the junctional and extrajunctional regions of normal, control pieces of muscle which had not been incubated with anti-neural cell adhesion molecule, they were lightly fixed, and incubated with the secondary gold-labelled antibody, before further fixation and processing for electron microscopy. Semi-thin, 0.5-micron sections of each were cut and viewed at 20,000 x magnification. Randomly sampled fibres from the extrajunctional regions of the denervated and innervated halves of the hemi-denervated gluteus and the normal, control gluteus were photographed for quantitative analysis. Five micrographs were randomly selected from each group and in these, the numbers, density and position of gold particles were measured using a digitizing tablet. By far the highest number of gold particles was found in the denervated half of the gluteus muscle, there being much less in the innervated half and practically none in the control. The density of gold particles in the denervated muscles was maximal in the plasma membrane-basal lamina complex, but most were located in the extracellular spaces outside this region, where they usually occurred in clusters apparently in association with collagen fibres. We conclude that neural cell adhesion molecule in denervated skeletal muscle is present not only in the plasma membrane but also in the basal lamina and extracellular space.

Animals↗

The effects of hypercapnia on intracellular and extracellular acid-base status in the toad Bufo marinus.

Toads (Bufo marinus) were exposed to environmental hypercapnia of 5% CO2 in air, and extracellular and intracellular acid-base parameters were determined 1 and 24 h after the onset of hypercapnia. The initial drop in pH was compensated by the elevation of extracellular and intracellular bicarbonate. Relating the pH compensation to the pH drop that is expected to occur by increased PCO2 at constant bicarbonate concentration, the pH compensation in the extracellular space was 30% and reached the following values for intracellular body compartments: 65% in skeletal muscle, 77% in heart muscle and 44% in skin. The additional bicarbonate was partly produced by blood and intracellular non-bicarbonate buffers; the major portion of the remainder was related to the excretion of ammonia into the environmental water. The hypercapnia-induced changes of pH were considerably smaller in all tissue cells than in the extracellular space. Thus Bufo marinus exhibits the relative preference of intracellular over extracellular acid-base regulation that has been observed in other vertebrates.

Acid-Base Equilibrium↗

Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusta migratoria) at rest and during flight.

Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoproteins epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.

Animals↗

[Effect of metabolic stress on the release of glutamic acid and GABA in the brain tissue of Mongolian hamsters].

The concentrations of glutamic acid and GABA were determined in the brain tissue in gerbils under conditions simulating "metabolic stress", that is ischaemia, aglycaemia and anoxia. The material for the determinations was taken from fragments of the hippocampus incubated under these conditions in artificial cerebrospinal fluid, and the concentrations of these neurotransmitters were determined by histochemical methods in vitro. The release of glutamic acid and GABA into the extracellular space increased with longer duration of the incubation in a linear fashion in all experimental groups and was most pronounced in ischaemia. In case of calcium absence in the extracellular space inhibition was observed of the release of these neurotransmitters which suggested an important role of bivalent cations in the regulation of the studied process, especially under control conditions. During anoxia and ischaemia a considerable part of the release of glutamic acid and GABA seems to be calcium-independent which may suggest presence of additional sources of release of the amino acid neurotransmitters, apart from their release from the direct pool. It is possible that these sources are activated during metabolic stress involving nerve cells.

Animals↗

Influences of experimental uremia on the intra- and extracellular acid-base status of the rat.

In an experimental study the influences of beginning uremia were studied in nephrectomized rats. It was observed: (1) Though there was found a continuous decrease of extracellular pH due to accumulation of fixed acids there was a slight increase of intracellular pH resulting from concomitant hypocapnia. (2) There was found a constant loss of bicarbonate from the extra- and intracellular body compartment which was much more pronounced for the extracellular space. This loss of bicarbonate reflects the progressing metabolic acidosis and demonstrates that the intracellular compartment is more protected against an increase of hydrogen ion concentration than the extracellular space is. (3) The stability of the intracellular acid-base status during uremic metabolic acidosis arises the question whether our clinical practice with alkali substitution according to the Mellemgard-Astrup equation is still adequate in these cases. (4) Blood-gas analysis can only give information about the acid-base status of the extracellular body compartment. The large intracellular space which is the aim of our clinical therapy is excluded from these measurements. Simple reliance of blood-gas analysis may lead to wrong conclusions and mistakes in therapy.

Acid-Base Equilibrium↗

Glycosphingolipid accumulation in the aortic wall is another feature of human atherosclerosis.

High accumulation of lipids is a typical feature of an atherosclerotic lesion. We have previously identified the chemical structure of the major glycosphingolipids (GSLs) of human aorta; however, quantification of the absolute concentration of GSLs was not carried out. In the present study, for the first time we have performed a quantitative comparative analysis of GSL composition in the media and two sublayers of the intima taken from normal regions, fatty streaks, and atherosclerotic plaques of the human aorta. The intimal tissue containing fatty streaks and atherosclerotic plaques accumulated GSLs, predominantly glucosylceramide (GlcCer), lactosylceramide (LacCer), and ganglioside GM3. GSL levels in plaques were highest: GlcCer was 18- and 8-fold, LacCer was 8- and 7-fold, and GM3 was 2.5- and 12-fold higher than in musculoelastic and elastic-hyperplastic intimal layers of normal regions, respectively. We did not observe a significant increase in other GSLs. An increase in the content of gangliosides GD3 and GD1a was detected in the media underlying atherosclerotic lesions. On the basis of an analysis of the ratio of GlcCer, LacCer, and GM3 accumulated in the tissue and cells of the elastic-hyperplastic layer of intima, we have concluded that the accumulation of the above-mentioned GSLs occurs mainly in the extracellular space of the intima. In this study, we have also demonstrated that extracellular lipid liposomes, which appear in the early stages of atherogenesis, are one locus of GSL accumulation in the extracellular space of the intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗