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PubMed1975

The ultrastructure of normal and glycerol treated muscle in the ghost crab, Ocypode cursor.

The ultrastructure of normal and glycerol treated fibers of the closer muscle of the ghost crab, Ocypode cursor, was studiedmthe muscle is composed of presumably phasic (short sarcomeres) and tonic (long sarcomeres) fibers, the latter greatly predominating. Horseradish peroxidase (HRP) was used as an extracellular tracer to delineate the tubular system (TS), and to determine to what extent this system becomes detached from the extracellular space as a result of glycerol treatment. Sarcolemmal clefts invade deeply into the muscle at Z-lines and I-bands; tubules invaginate into the muscle from the clefts and from the surface sarcolemma at the Z-lines, A-I overlaps and A-bands. A tubules are in frequent diadic or tetradic contact with the sarcoplasmic reticulum (SR), whereas Z tubules appear to be randomly associated with SR, terminal cisterns (TC) and Z-line fibrils. When HRP was administered to normal muscle, black reaction product was found adjacent to the outer surface of the sarcolemma, within the clefts and within profiles of the TS throughout the tissue. In glycerol treated muscle peripheral vacuolation frequently occurred; black reaction product penetrated only as far as the vacuoles and into dilated Z-line tubules, but was virtually absent from the rest of the TS. This lack of continuity between the extracellular space and the A tubules indicated disruption or constriction of the A tubules as a result of glycerol treatment, although Z tubule contact with the extracellular space appeared unimpaired. These findings provide ultrastructural correlates of the electrophysiological changes produced by glycerol treatment of the closer muscle of the ghost crab (Papir, 1973), namely, interference with excitation-contraction (e-c) coupling. The random association of the Z tubules with SR and TC, and their resistance to disruption by glycerol treatment, tend to endorse the claims that the Z tubules in crustacean muscle are not directly involved in e-c coupling (Brandt et al., 1965; Peachey, 1967; Selverston, 1967).

Animals↗
PubMed1992

Effects of light and darkness on pH outside rod photoreceptors in the cat retina.

We recorded pH in the extracellular space surrounding rod photoreceptors in the dark-adapted eye of the cat and during illumination with double-barreled H(+)-selective microelectrodes. A pH of 7.17 was recorded in the vitreous at the retinal surface of the dark-adapted eye and this became more alkaline during light adaptation. In dark adaptation, a pH close to 7.00 was recorded in a region of maximal acidity in the extracellular space surrounding rods in the outer nuclear layer (ONL). pH steeply alkalinized as the microelectrode was moved more distally towards the retinal pigment epithelium (RPE), and almost reached the pH of the arterial blood at the apical surface of the RPE. Illumination produced an intraretinal alkalinization that was largest (up to 0.2 pH units) in the ONL, maximal in amplitude at rod-saturating intensities, and that was sustained during steady background illumination. The light-evoked alkalinization was relatively slow in onset, having a time constant (1/e) of 64 sec, and took 8-12.5 min to return to the dark-adapted level after the offset of maintained illumination. These results show that acid production by cat rods is highest in the dark, reflecting a high rate of energy metabolism, and suggest that glycolysis is required to support the dark current. Illumination, by suppressing both glycolysis and respiration, alkalinizes the extracellular space surrounding rods. The substantial change in pH outside rods from dark to light could alter pH dependent properties of the interphotoreceptor matrix.

Adaptation, Ocular↗
PubMedDate not supplied

An analysis of 45Ca fluxes in the rabbit lens.

The uptake of 45Ca by the rabbit lens reached equilibrium within 20 hr; the steady state lens/medium ratio was 0.08. This ratio is similar to that found for the lens/medium distribution of extracellular markers. Efflux of 45Ca from the lens was rapid and insensitive to iodoacetate. Mathematical analysis of 45Ca efflux curves revealed that calcium efflux from the lens could be described solely upon the basis of passive movement from the extracellular space. It is concluded that the exchangeable calcium in the normal rabbit lens is predominantly located in the extracellular space.

Animals↗
PubMed1995

Sequestration of acetylated LDL and cholesterol crystals by human monocyte-derived macrophages.

Monocyte-derived macrophages accumulate and process cholesterol in atherosclerotic lesions. Because of the importance of this process, we examined the interaction of cholesterol crystals and acetylated low density lipoprotein (AcLDL) with human monocyte-macrophages in a combined chemical and morphological study. These two forms of cholesterol induced extensive compartmentalization of the macrophage cytoplasm. Unexpectedly, the compartments maintained a physical connection to the extracellular space as demonstrated with ruthenium red staining. The compartments formed through invagination of the top surface of the macrophage plasma membrane. Some cholesterol crystals and AcLDL were sequestered within these surface-connected compartments for up to five days in the case of the crystals and for one day in the case of AcLDL. Pulse-chase studies of fractionated macrophages indicated that [3H]cholesterol redistributed from the surface-connected compartments into lysosomes (where the cholesterol remained unesterified) and into lipid droplets (where the cholesterol was stored as cholesteryl ester). Intracellular uptake and esterification of cholesterol was blocked by cytochalasin D. However, once cholesterol was sequestered in the surface-connected compartments, subsequent esterification of the cholesterol could not be inhibited by cytochalasin D. Apolipoprotein E was localized within the surface-connected compartments by immunogold labeling suggesting a possible function for this protein in the processing of lipid taken up through the sequestration pathway. Removal of microcrystalline cholesterol from the medium resulted in release of most of the accumulated cholesterol microcrystals from the macrophages, as well as disappearance of the surface-connected compartments. Thus, sequestration is a novel endocytic mechanism in which endocytic compartments remain connected to the extracellular space. This differs from phagocytosis where endocytic vacuoles rapidly pinch off from the plasma membrane. Sequestration provides a means for macrophages to remove substances from the extracellular space and later release them.

Apolipoproteins E↗
PubMed1984

Neuronal interactions mediated by neurally evoked changes in the extracellular potassium concentration.

Neuronal interactions mediated by alteration of the extracellular K+ concentration [K+]o occur between populations as well as among single neurones in very restricted regions. The interactions mediated by K+ ions may range from low efficacy ones (in which the effects of increased [K+]o around the non-active cells can be recorded only after massive activity of a large population of neurones) to very effective interactions (in which a single action potential in a neurone is sufficient to produce a depolarization of several mV in a second one). Such efficient K+-mediated interactions cannot be unequivocally distinguished by shape, amplitude or time course from postsynaptic responses induced by chemical or electrotonic synapses. We review here experiments which demonstrate various levels of interactions mediated by changes in potassium ion concentration. The giant axons (Gax) and non-giant axons from the central nervous system of the cockroach Periplaneta americana were used. The types of interactions discussed are: pathological interactions among populations of neurones induced by the convulsant drug picrotoxin; restricted and limited interactions which are the consequence of the combination of the special geometry of Gaxs and increases in extracellular K+; and finally, local and efficient interactions among Gaxs which are postulated to be mediated by K+ ions. The experiments described in this review, as well as others, demonstrate that the extracellular spaces in the CNS serve as predetermined pathways for K+-mediated neuronal communication. When the extracellular space between two adjacent neurones is very small, the K+-mediated interaction may resemble the PSPs of chemical or electrotonic synapses. It is possible that because of this resemblance, other K+-mediated interactions in the CNS have not been identified as such.

Abdomen↗
PubMed1985

Diffusion from an injected volume of a substance in brain tissue with arbitrary volume fraction and tortuosity.

When a substance is pressure-injected from a micropipette into the extracellular space of the brain it may either form a cavity or it may infiltrate the extracellular space. In either case subsequent diffusion is governed by the volume fraction and tortuosity of the brain tissue as well as the diffusion coefficient of the substance itself. Appropriate equations, solutions and approximations to these problems are discussed. The results are relevant to the interpretation of studies on neuropharmacology and in situ electrochemistry.

Brain↗
PubMed2003

Establishment of cadherin-based intercellular junctions in the dermal papilla of the developing hair follicle.

During hair follicle development, mesenchymal cells aggregate to form the dermal papilla with hair-inducing activity. However, the cellular mechanisms underlying the aggregative behavior of dermal papilla cells are less known. The present study demonstrates that cadherin-based intercellular junctions interconnect dermal papilla cells in developing hair follicles of mice. It is shown that as mesenchymal cells aggregate to be surrounded by epithelium in developing hair follicles, cadherin-11 comes to exhibit the dotted patterns of distribution. The appearance of the dot-like distribution of the molecule is concomitant with the formation of intercellular junctions in the mesenchymal aggregate, which make a tightly packed population of cells with little extracellular space. At later stages of the development, although extracellular space reappears in the dermal papilla, the cells remain interconnected by well-developed intercellular junctions, where cadherin-11 as well as beta-catenin is localized. Taking into consideration the normal hair development in cadherin-11 mutant mice, it might be that multiple cadherins are responsible for the establishment of intercellular junctions in the dermal papilla and serve to maintain the aggregative behavior of the cells.

Animals↗
PubMed1991

Folding and function of the myelin proteins from primary sequence data.

To explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular. In this program, propensities for the secondary structure conformations as well as physical-chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two-dimensional map of the correlation coefficients. Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that the conserved residues included most of the amino acids which were predicted to form the alpha or beta conformations, while the altered residues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of beta propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed alpha-beta-alpha folding. To trace the immunoglobulin fold along the P0 sequence, we compared the beta propensity curve of P0 with that of the immunoglobulin M603, whose three-dimensional structure has been determined. We propose that the flat beta-sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra beta-fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces and may account for a smaller extracellular space as measured by x-ray diffraction. A computer search of the myelin protein sequences for functional motifs revealed sites for N-glycosylation, phosphorylation, nucleotide binding, and certain enzyme activities. We note especially that there are potential nucleotide binding sites in proteolipid protein (PLP), MBP and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). This is consistent with the experimental observations that PLP acts like an ionophore or proton channel when reconstituted into planar lipid bilayers, MBP binds GTP, and CNP catalyzes in vitro the hydrolysis of 2',3'-nucleotides into corresponding 2'-nucleotides.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1975

Transport of choline from plasma to cerebrospinal fluid in the rabbit with reference to the origin of choline and to acetylcholine metabolism in brain.

The supply of choline to the brain and the interrelationship between acetylcholine metabolism in the brain, choline in cerebrospinal fluid (CSF) and choline in plasma has been investigated by studying the transport of deuterium labelled choline from plasma toCSF in rabbits. The experiments were performed either by i.v. infusion of labelled choline or by administering deuterium choline in the diet until steady state levels were reached. All assays of labelled as well as unlabelled choline and acetylcholine were carried out by gas chromatographic-mass spectrometric technique. From these experiments it was found that the half-lives of the disappearance of labelled choline in plasma and CSF were similar (15 min) and the mole fraction of labelled choline in plasma was approximately 2.4 times that in CSF. The half-life of labelled acetylcholine in brain was about 25 min. In the time interval 10-20 min, the mole fraction of brain acetylcholine was the same as that of CSF choline. When infusion was carried out during pentobarbital anaesthesia the mole fraction of labelled brain acetylcholine decreased and that of labelled CSF choline increased compared to those of conscious animals. At steady state the mole fraction of labelled choline in plasma was nearly twice as high as that in CSF which in turn was significantly higher than that of acetylcholine in the brain. From these results it is suggested that in rabbit, free and derived choline in plasma are supplied to the brain in equal amounts. Furthermore it is suggested that choline is transported from plasma, via the extracellular space of the brain to CSF. In the extracellular space a part of choline is diverted and exchanged with choline in the acetycholine metabolic pathway.

Acetylcholine↗
PubMed1995

Investigation into the role of N-acetylaspartate in cerebral osmoregulation.

Marked abnormalities of the magnetic resonance intensity of N-acetylaspartate (NAA) have been reported in patients with various neurological disorders, but the neurochemical consequences of these alterations are difficult to assess because the function of NAA remains speculative. The purpose of this study was to examine whether NAA plays a role in protecting neurons against osmotic stress. Intracerebral microdialysis was used to expose a small region of the rat dorsolateral striatum to an increasingly hyposmotic environment and to measure resulting changes in NAA extracellular concentrations. NAA changes in the extracellular fluid (ECF) were compared with those of the amino acids, in particular, taurine, known to be involved in brain osmoregulation. Stepped increases in cellular hydration produced by hyposmotic perfusion media induced a marked increase in ECF NAA, reflecting a redistribution of NAA from intra-to extracellular space. Parallel experiments showed that, of all the extracellular amino acids measured, only taurine markedly increased with hyposmolar perfusion medium, indicating that the ECF NAA increase associated with hyposmotic stress was a specific response and not passive leakage out of the cells. As NAA is predominantly neuronal, it may contribute to the protection of neurons against swelling (i.e., regulatory volume decrease). In conditions with impaired blood-brain barrier and cytotoxic oedema, efflux of intracellular NAA subsequent to sustained cellular swelling might lead to a reduction in total brain NAA detectable by magnetic resonance spectroscopy. Alternatively, redistribution of NAA from intra-to extracellular space implies changes in its chemical environment that may alter its magnetic resonance visibility.

Amino Acids↗
PubMed2006

S100B binding to RAGE in microglia stimulates COX-2 expression.

Besides exerting regulatory roles within astrocytes, the Ca2+-modulated protein of the EF-hand type S100B is released into the brain extracellular space, thereby affecting astrocytes, neurons, and microglia. However, extracellular effects of S100B vary, depending on the concentration attained and the protein being trophic to neurons up to nanomolar concentrations and causing neuronal apoptosis at micromolar concentrations. Effects of S100B on neurons are transduced by receptor for advanced glycation end products (RAGE). At high concentrations, S100B also up-regulates inducible NO synthase in and stimulates NO release by microglia by synergizing with bacterial endotoxin and IFN-gamma, thereby participating in microglia activation. We show here that S100B up-regulates cyclo-oxygenase-2 expression in microglia in a RAGE-dependent manner in the absence of cofactors through independent stimulation of a Cdc42-Rac1-JNK pathway and a Ras-Rac1-NF-kappaB pathway. Thus, S100B can be viewed as an astrocytic endokine, which might participate in the inflammatory response in the course of brain insults, once liberated into the brain extracellular space.

Animals↗
PubMed1996

Hemodynamic characteristics of extracellular UTP in the perfused rat liver.

Uridine 5'-triphosphate (UTP) is stored in the granules of cells such as platelets and is released into the extracellular space upon cell stimulation. Extracellular UTP is known to influence many biological processes. We investigated the hemodynamic effects of UTP on the perfused rat liver and characterized its receptors. Liver perfusions were performed in a recirculation system under constant pressure (28 cmH2O). The perfusion flow and oxygen consumption rate were measured at 30 second intervals. UTP decreased the perfusion flow and the oxygen consumption rate, dose-dependently. UTP-induced changes were transient and disappeared in about 10 minutes. Suramin (P2-purinergic antagonist, 100 uM) and indomethacin (cyclooxygenase inhibitor, 20 uM) blocked UTP-induced hemodynamic changes significantly. The effects of UTP were also inhibited when Kupffer cells were damaged with treatment of gadolinium chloride (10 mg/kg iv). L-NAME (1 mM), a potent inhibitor of nitric oxide synthase, markedly enhanced and prolonged the contractile response of UTP in the hepatic vessel. These results suggest that UTP acts mainly on suramin-sensitive UTP receptors on the Kupffer cell through prostanoid synthesis. The nitric oxide systems in the endothelium seem to counteract the vasoconstrictile action of UTP in the hepatic circulation.

Animals↗
PubMedDate not supplied

Structures in material transference and vitelline envelope formation in Betta splendens follicles.

Structures were found by transmission electron microscopy, they were located within follicular cells and the oocyte, and in the interspace between them in follicles of the teleost fish Betta splendens. Some structures with features characteristic or lamellar bodies were found in small follicles. The possible role of these structures in the formation of the vitelline envelope as well as in the material transference is discussed. Vacuoles, vesticles and particles intensely stained were found in the microvilli and the cortical cytoplasm of the oocyte at the onset of vitellogenesis. These results suggest that different substances present in the cellular components of the follicle might be transferred from cell to cell through the extracellular space and through the prolongations that cross the extracellular space.

Animals↗
PubMed1996

In vivo, continuous and automatic monitoring of extracellular ascorbic acid by microdialysis and on-line liquid chromatography.

A system for in vivo, automatic, continuous monitoring of organ extracellular ascorbic acid in anesthetized rat is described. This system involves microdialysis perfusion and a LC system equipped with an electrochemical detector. Microdialysate, eluted from a microdialysis probe implanted in the brain cortex or in the left ventricular myocardium of anesthetized rats was collected in the sample loop of an on-line injector for direct injection onto the LC system. This automated method provides a shortened sample processing time. This system was utilized to investigate the effect of cerebral ischemia on cortex extracellular ascorbic acid and the effect of myocardial ischemia on left ventricular myocardium extracellular ascorbic acid in anesthetized rats. Basal ascorbic acid concentrations in the cortex and left ventricular myocardium ranged from 9.7 to 15.4 microM (mean +/- S.D., 12.7 +/- 2.5 microM from the results of eight rats) and from 9.3 to 36.0 microM (mean +/- S.D., 24.3 +/- 8.9 microM from the results of twelve rats), respectively. Cerebral ischemia significantly elevated ascorbic acid levels in the cortex extracellular space, while myocardial ischemia did not significantly alter ascorbic acid levels in the left ventricular myocardium extracellular space.

Animals↗
PubMed1976

The effect of denervation on the synchronization of contraction of the rat vas deferens.

Denervation of the rat vas deferens results in both quantitative and qualtitative changes in the contraction of the isolated smooth muscle. The maximum contraction induced by norepinephrine, acetylcholine or serotonin is enhanced by denervation. The response of the tissue to 2 X 10(-4) M BaCl2 initiates a contraction which is more synchronized than the response of the control tissue. By use of 14C-sorbitol and Co-EDTA for estimating extracellular space and from fluorescence histochemical observations, it appears that facilitation of drug diffusion through the extracellular space plays an unimportant role in the altered response to drugs. In experiments which utilized a double-compartment tissue bath it was found that denervation enhanced the propagation of the contraction in the longitudinal direction. From measurements of the longitudinal tissue impedance it was determined that the resistance between smooth muscle cells was significantly decreased by denervation. These results suggest that denervation improves the propagation of excitation in this smooth muscle.

Animals↗
PubMed1998

Effects of acute glucose overload on histamine H2 receptor-mediated Ca2+ mobilization in bovine cerebral endothelial cells.

[Ca2+]i and whole-cell membrane current were measured in microvascular endothelial cells from bovine brain. The effects of histamine on [Ca2+]i were examined, and the acute effect of changing extracellular glucose concentration on Ca2+ homeostasis was investigated. Application of 10 micromol/l histamine evoked an initially transient and then sustained increase in [Ca2+]i in normal Krebs solution, but only the transient component in Ca2+-free solution, thereby indicating that histamine mobilizes Ca2+ both from intracellular store sites and extracellular space. The effects of histamine on [Ca2+]i were inhibited by the H2 antagonists, ranitidine and cimetidine, but not by the H1 antagonist, pyrilamine. Incubation of the cells for 2 h in solutions containing low (1.1 and 2.3 mmol/l) or high (23 mmol/l) concentrations of glucose did not influence the resting level of [Ca2+]i. Treatment with low concentrations of glucose did not impair histamine-induced Ca2+ mobilization. On the other hand, when histamine was applied to the cells pretreated with 23 mmol/l glucose, it failed to mobilize Ca2+ from both intracellular store sites and extracellular space. The effect of histamine was mimicked by dibutyryl cyclic AMP, but glucose overload failed to inhibit this, suggesting that glucose overload inhibits H2 receptor-mediated cyclic AMP production. Glucose overload-induced impairment of histamine action was reversed by pretreatment with staurosporine and calphostin C and mimicked by phorbol-12,13-dibutyrate, thereby suggesting the involvement of protein kinase C in the high glucose-induced inhibition of Ca2+ mobilization. Whole-cell membrane current measurement showed that there was no difference in the membrane currents between control and high glucose-treated cells. These results indicate that in bovine brain microvascular endothelial cells, histamine induces Ca2+ release from intracellular store sites and subsequent entry from the extracellular space through the activation of H2 receptors. Glucose overload acutely inhibits histamine-induced Ca2+ mobilization by the activation of protein kinase C.

Animals↗
PubMed2004

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants.

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.

Amine Oxidase (Copper-Containing)↗
PubMed1980

Sulfated proteoglycan accumulation during development of the embryonic chick limb bud studied by electron microscopic autoradiography.

Cartilage cells are characterized by a high concentration of extracellular sulfated proteoglycan. Electron microscopic autoradiography was used to compare the incorporation of sulfate into proteoglycan by limb-bud chondrogenic and myogenic cells. From stage 19 to stage 21 there was no significant difference between the cartilage- and muscle-forming regions in the number of silver grains over either the extracellular space or the intracellular space. From stage 22 to 25 the number of extracellular silver grains was significantly greater in the chondrogenic region than in the myogenic region, but the number of intracellular silver grains was the same. Since most of the silver grains were intracellular, no significant difference in the total number of grains was found between the two tissues. Stage-26 and -27 embryos showed a significantly greater number of silver grains over both the cells and the extracellular space in the cartilage region than in the muscle region. Thus, the first step of cartilage differentiation involves a decrease in the extracellular deposition of sulfated proteoglycan in the myogenic region rather than an increase in deposition in the chondrogenic region between stage 22 and 25. After stage 25 there is an increase in sulfated proteoglycan synthesis in the chondrogenic region to the myogenic region.

Animals↗