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At least 1,765 records · Page 98Linked to original sources
PubMed1999

Platelet-activating factor induced Ca(2+) signaling in human microglia.

Increases in intracellular Ca(2+) concentration in human microglial cells in response to platelet-activating factor (PAF) were studied using Ca(2+)-sensitive fluorescence microscopy. In normal physiological solution (PSS), PAF-induced transient increases in [Ca2+](i) which recovered to baseline values within 200 s. Application of PAF in zero-Ca(2+) solution caused the peak response to be decreased to a value near 20% of that recorded in PSS suggesting a primary contribution of Ca(2+) influx for the [Ca2+](i) increase in PSS. To investigate PAF-induced Ca(2+) influx, the contents of intracellular stores were modulated using the SERCA blocker cyclopiazonic acid (CPA). The Ca(2+) signal induced by CPA (10 microM) in zero-Ca(2+) solution showed a peak response about 20% of the amplitude in the presence of external Ca(2+), suggesting the latter response included significant contributions from store-operated Ca(2+) entry. The influx of divalent cations with PAF or CPA was directly measured using Mn(2+) quenching of the fluorescence signal. Although both PAF and CPA induced a similar degree of Mn(2+) influx over time, the PAF effect was very rapid, whereas the CPA action was delayed and only evident about 200 s after application. Overall, the results show that the primary source of the PAF-induced increase of [Ca2+](i) in human microglia was the influx of Ca(2+) from the extracellular space and intracellular Ca(2+)-release contributed only a small part of the total Ca(2+) signal. Nevertheless, Ca(2+)-release induced by PAF (or CPA) serves as an important factor in controlling Ca(2+) entry presumably mediated by activation of store-operated-Ca(2+) channels.

Calcium↗
PubMed1978

Anatomy of germinal centers in mouse spleen, with special reference to "follicular dendritic cells".

Lymphocyte proliferation in germinal centers (GC's) is thought to be triggered by antigen retained extracellularly on the surface of special "dendritic" cells. The anatomy and function of these cells have not been studied directly or in detail. We therefore examined mouse spleen GC's developing in response to sheep erythrocyte stimulation. We found that distincitve "follicular dendritic cells" (FDC's) were present in both the GC and adjacent mantle region of secondary follicles. The large, irregularly shaped nucleus, containing little heterochromatin, allowed for the light microscope (LM) identification of FDC's. By EM, the cell was stellate in shape sending out long, thin sheets of cytoplasm which could fold and coil into complex arrays. The processes were coated extracellularly by an amorphous electron-dense material of varying thickness, as well as particulates including variable numbers of virions. The FDC cytoplasm lacked organelles of active secretory and endocytic cells, such as well-developed rough endoplasmic reticulum (RER) and lysosomes. These anatomical features readily distinguished FDC's from other cell types, even those that were extended in shape. To pursue these descriptive findings, we injected three electron-dense tracers i.v. and sacrificed the mice 1 h-10 days thereafter. Colloidal carbon, colloidal thorium dioxide (cThO2), and soluble horseradish peroxidase (HRP) were actively sequestered into the vacuolar system of macrophages but were interiorized only in trace amounts by FDC's. Therefore, FDC's are not macrophages by cytologic and functional criteria. FDC's did display a unique property. Both colloidal carbon and thorium dioxide, which are nonimmunogens, could be visualized extracellularly on the cell surface for several days. The meaning of this is unclear, but the association of colloid with FDC's appeared to slow the movement of particulates through the extracellular space into the GC proper. FDC's were not readily identified in splenic white pulp lacking GC's. They must develop de novo then, possibly from novel dendritic cells that we have identified in vitro (Steinman, R. M., and Z. A. Cohn. 1973. J. Exp. Med. 137:1142-1162).

Animals↗
PubMed1985

The distribution of serum albumin in the rat testis, studied by electron microscope immunocytochemistry on ultrathin frozen sections.

The distribution of serum albumin is of interest in the rat testis because this protein is the principal carrier for testosterone in the plasma and interstitial fluid of this species. We have localized extravascular serum albumin in the rat testis at the electron microscope level, using gold particle immunocytochemistry on ultrathin frozen sections of tissue fixed lightly by perfusion. The same localization was obtained with three different antisera. Preabsorption and normal rabbit serum controls were negative, and Western blots of testis extracts showed major activity only at the molecular weight of albumin. Serum albumin occurred in substantial concentration throughout extracellular space in the interstitial tissue, as well as in the space between the boundary layer and the base of the seminiferous epithelium. Immunoreactivity extended between Sertoli cells, as well as around spermatogonia and early primary spermatocytes (to stage 11), but did not traverse the Sertoli-Sertoli junctions that comprise the blood-testis barrier. Macrophages in the interstitial tissue showed some endocytic activity. If perfusion fixation was carried out in a manner that flushed most of the albumin from the interstitial space, then a layer of albumin remained on the surface of Leydig cells and many macrophages but was minimal or absent on the surface of other cell types that are normally in contact with albumin, such as Sertoli cells, spermatogonia, myoid cells, lymphatic endothelium, fibroblasts, or cells of blood vessels.

Animals↗
PubMed2000

Mechanism of ATP-induced [Ca(2+)](i) mobilization in rat basilar smooth muscle cells.

BACKGROUND AND PURPOSE: We have previously reported that extracellular ATP activates P(2u) receptors and increases intracellular free Ca(2+) ([Ca(2+)](i)) by G protein/phospholipase C/inositol 1,4,5-triphosphate pathways in cerebral artery smooth muscle cells. However, the possible contribution of other signaling pathways remains unclear. This study was undertaken to investigate the role of protein tyrosine kinase (PTK) and mitogen-activated protein kinase (MAPK) in mediating ATP-induced Ca(2+) mobilization in rat basilar artery smooth muscle cells (RBASMCs). METHODS: RBASMCs were freshly isolated, and [Ca(2+)](i) was monitored by fura 2 microfluorimetry. MAPK phosphorylation was studied by the Western blot technique. RESULTS: ATP produced a biphasic [Ca(2+)](i) response, which consists of releasing Ca(2+) from internal stores and influx from extracellular space. PTK inhibitors tyrphostin 51 and genistein inhibited [Ca(2+)](i) response to ATP. Tyrphostin A1, an inactive analogue of tyrphostins, failed to reduce the ATP-induced response. MAPK kinase inhibitor PD98059, but not U0126, reduced the ATP-induced [Ca(2+)](i) response. Phosphatidylinositol 3-kinase (PI3-K) tyrosine kinase inhibitor wortmannin, but not janus tyrosine kinase (JAK2) inhibitor AG490, partially inhibited the [Ca(2+)](i) response induced by ATP. In addition, ATP enhanced MAPK phosphorylation in a concentration- and time-dependent manner, and genistein, tyrphostin 51, PD98059, and U0126 inhibited MAPK phosphorylation. CONCLUSIONS: Extracellular ATP produced [Ca(2+)](i) elevation and MAPK phosphorylation in RBASMCs, and the effect was regulated by PTK. The role of MAPK in ATP-induced [Ca(2+)](i) elevation is not clear. PI3-K tyrosine kinase and JAK2 tyrosine kinase may not play an important role in the ATP-induced [Ca(2+)](i) response in RBASMCs.

Adenosine Triphosphate↗
PubMed1984

The permeability of the blood-brain barrier in mice suffering from fatal lymphocytic choriomeningitis virus infection.

The ultrastructure and the blood-brain-barrier (BBB) permeability were studied in mice suffering from lymphocytic choriomeningitis (LCM). Brains and meninges from mice suffering from LCM virus-induced lymphocytic choriomeningitis were studied by investigating the BBB function and by electron and light microscopy. The cellular exudate in the leptomeninges was located in the subarachnoid space, in arachnoidea and pia, and it was dominated by proliferated pial cells and mononuclear cells, most of which were lymphocytes, while there were only a few neutrophil granulocytes. Many intravascular lymphocytes were seen adhering to as well as penetrating the vessel walls. Many of these lymphocytes were morphologically compatible with T cells. Lymphocytes and larger mononuclear cells were also accumulated in the choroid plexus, and lymphocytes were present in the ventricular system with a tendency to adhere to ependymal epithelial cells. Inspection of the ultrathin sections incubated for horseradish peroxidase (HRP)-activity revealed that the overwhelming part of the peroxidase activity was localized in the extracellular space of the meningeal vessel walls and especially in the abundant intercellular fluid which, like the inflammatory cells, was found in the subarachnoid space in arachnoidea and in pia. In the neuropil, only very small quantities of reaction product were seen intercellularly in the most superficial layers of the cortex. The tight junctions were always intact, but the possibility of a non-demonstrable opening is discussed. Evaluation of the BBB permeability for 2-amino[1-14C]isobutyric acid (AIB) was made by quantitative autoradiography, and it was demonstrated convincingly that AIB concentrations in the subpial and perichorodial tissues were markedly increased in diseased animals as compared to the controls. Our results seem to contradict previous theories on the cause of death resulting from the LCM disease. The findings presented here do not speak in favor of a pronounced brain edema, just as results obtained by us and others do not speak for the possibility of the death being caused by convulsive seizures with subsequent brain anoxia. However, our observations are compatible with the hypothesis that cytotoxic T cells may interact in vivo with virus-infected targets, which are essential for the regulation of the composition of the cerebrospinal fluid. On the other hand, the dysfunction of the BBB demonstrated adds a new element to the pathologic mechanism in a model for the study of virus-induced meningitis.

Animals↗
PubMed1984

Improvement in injury induced hypocalcia by high-dose naloxone intervention.

Naloxone hydrochloride was used to re-establish normal calcium activities in the extracellular space of the injured spinal cord. The micromolar levels of calcium which occur immediately after injury were improved above control values by 1 h 30 min post-injury; normal activities occurred at 2 h 45 min. Such a restitution of ionic levels after injury may contribute to the beneficial effects of naloxone in traumatic injury.

Animals↗
PubMed1967

Regulation of cardiac muscle contractility.

The heart's physiological performance, unlike that of skeletal muscle, is regulated primarily by variations in the contractile force developed by the individual myocardial fibers. In an attempt to identify the basis for the characteristic properties of myocardial contraction, the individual cardiac contractile proteins and their behavior in contractile models in vitro have been examined. The low shortening velocity of heart muscle appears to reflect the weak ATPase activity of cardiac myosin, but this enzymatic activity probably does not determine active state intensity. Quantification of the effects of Ca(++) upon cardiac actomyosin supports the view that myocardial contractility can be modified by changes in the amount of calcium released during excitation-contraction coupling. Exchange of intracellular K(+) with Na(+) derived from the extracellular space also could enhance myocardial contractility directly, as highly purified cardiac actomyosin is stimulated when K(+) is replaced by an equimolar amount of Na(+). On the other hand, cardiac glycosides and catecholamines, agents which greatly increase the contractility of the intact heart, were found to be without significant actions upon highly purified reconstituted cardiac actomyosin.

Adenosine Triphosphatases↗
PubMed1995

Biochemical analysis of starch degradation by Ruminobacter amylophilus 70.

Ruminobacter amylophilus is an obligate anaerobe that uses only alpha-linked glucose molecules (i.e., maltose, maltodextrins, and starch) as a source of energy, making it an excellent model for the study of bacterial starch degradation. Constitutive amylase, amylopectinase, and pullulanase activities were found in intracellular and extracellular fractions of R. amylophilus. However, extracellular activities apparently resulted from cell lysis. Both soluble and membrane-bound polysaccharidase activities were detected. Most of the soluble polysaccharidase activity partitioned with the periplasmic cell fraction. No alpha-glucosidase or maltase activity was detected in either the cellular or extracellular fraction. In addition, intact cells of R. amylophilus bound U-14C-starch. This binding could be saturated and was constitutive and sensitive to proteinase K, indicating protein or protein complex mediation. Competition experiments showed that these starch-binding sites had equally high affinities for starch and maltodextrins larger than maltotriose. The sites had a reduced affinity for maltose and virtually no affinities for glucose and nonstarch polysaccharides. These findings suggest that R. amylophilus binds starch molecules to the cell surface as an initial step in transporting the molecule through the outer membrane and into the periplasmic space. Extracellular polysaccharides do not appear to be involved in starch degradation.

Amylases↗
PubMed1976

Lymphocyte emigration from high endothelial venules in rat lymph nodes.

Sequential events during lymphocyte emigration from high endothelial venuses (HEV) were studied by scanning and transmission electron microscopy combined with regional perfusion techniques. The results indicate that blood lymphocytes selectively adhere to HEV surfaces through microvilli which attach to shallow pits on the luminal surfaces of high endothelial cells. These intercellular contact points resist hydrodynamic and osmotic shearing forces, but can be disrupted by treatments which remove endothelial glycocalyx, hydrolyse lymphocyte surface glycoproteins, or chelate divalent cations. After this initial attachment phase, lymphocytes enter apical clefts between endothelial cells where they assume a motile configuration characterized by loss of microvilli and formation of irregular surface folds. Intramural lymphocytes adhere to adjacent endothelial cells through macular and villous contacts. Fibrillar electron-dense material traverses the 15-20 nm gap at these points of adhesion. Microtubules and microfilaments are also seen around areas of cytoplasmic constriction in these motile lymphocytes. The migrating lymphocytes show cytoplasmic polarity which is oriented in the direction of movement as they pass through extracellular spaces in the venular wall and cross successive laminations in the perivascular sheath to enter the node. Since these lymphocytes enter channels between endothelial cells which are stained by intralymphatic injections with horseradish peroxidase, it is suggested that their entry into the node depends upon migration along a chemotactic gradient.

Animals↗
PubMed1984

Water exchange in plant tissue studied by proton NMR in the presence of paramagnetic centers.

The proton NMR relaxation of water in maize roots in the presence of paramagnetic centers, Mn2+, Mn- EDTA2 -, and dextran-magnetite was measured. It was shown that the NMR method of Conlon and Outhred (1972, Biochem. Biophys. Acta. 288:354-361) can be applied to a heterogenous multicellular system, and the water exchange time between cortical cells and the extracellular space can be calculated. The water exchange is presumably controlled by the intracellular unstirred layers. The Mn- EDTA2 - complex is a suitable paramagnetic compound for complex tissue, while the application of dextran-magnetite is probably restricted to studies of water exchange in cell suspensions. The water free space of the root and viscosity of the cells cytoplasm was estimated with the use of Mn- EDTA2 -. The convenience of proton NMR for studying the multiphase uptake of paramagnetic ions by plant root as well as their transport to leaves is demonstrated. A simple and rapid NMR technique (spin-echo recovery) for continuous measurement of the uptake process is presented.

Edetic Acid↗
PubMed1986

The intracellular and extracellular changes associated with ischaemia--effects of catecholamines in arrhythmogenesis.

Beta sympathetic stimulation and the beta-adrenergic agonists exert a variety of electrophysiologic effects which contribute to the genesis of ischaemia-related arrhythmias. These are both primary, most likely related to enhancement of the slow calcium current, and secondary, resulting from changes in the metabolism of the ischaemic myocardium as reflected in the accumulation of ions and other end products of metabolism in the extracellular space. In addition, the regional influence of these changes may contribute to the ionic and electrical inhomogeneities. Further studies are needed to define more precisely the factors responsible for the enhanced arrhythmogenicity attributed to beta-adrenergic stimulation.

Adrenergic beta-Agonists↗
PubMed1971

Comparison of glycerol treatment in frog skeletal muscle and mammalian heart. An electrophysiological and morphological study.

Frog skeletal muscle and mammalian heart muscle were studied in vitro before and after glycerol treatment. Loss of contractility, changes in the action potential and disruption of the T system were observed in skeletal muscle cells. In mammalian heart muscle the T system was not disrupted with hypertonic glycerol treatment, and no significant electrophysiological changes were observed. The continuity between the T system and the extracellular space was investigated by diffusion tracer methods. Decrease of contractility during the hypertonic phase in the glycerol treatment was found to depend on tonicity. The results of this study clearly show that not only are there differences in morphology between skeletal and cardiac muscle, but there are also differences in the resistance to osmotic changes.

Action Potentials↗
PubMed1975

Myosin in developing normal and dystrophic chicken pectoralis. II. The relationship between intracellular and extracellular aspartate pools and myosin synthesis.

The present studies establish the cellular pool of aspartate as the major source of this amino acid used during the biosynthesis of skeletal muscle myosin. The precursor=product relationship has been established in growing, normal pectoralis and has been suggested in hypertrophied, dystrophic pectoralis. Specific activities of aspartic acid in plasma and cellular pools corrected for extracellular space contributions have been correlated with aspartate incorporation onto myosin. These data have been coupled with quantitative data on myosin accumulation and have established the cellular pool as the major precursor pool. The data also give further insight into some of the factors responsible for the observation that in vivo dystrophic tissue gives higher levels of aspartate incorporation than normal tissue.

Animals↗
PubMed1978

The cells of the rat gastric groove and cardia. An ultrastructural and carbohydrate histochemical study, with special reference to the fibrillovesicular cells.

The distal wall of the groove between the rat forestomach and glandular stomach is lined with a special type of columnar cells (CCGG) and with fibrillovesicular cells (FVC). The cardiac glands contain cardiac mucosa (CMC) and serous cells (CSC). The CCGG contain small mucous granules and special vesicles and tubules. The CMC are filled with large mucous granules and resemble mucous neck cells. The CSC are filled with large proteinaceous granules. The FVC are characterized by long microvilli, apical bundles of microfilaments and a complex "tubulovesicular system". The pattern of 3H-thymidine incorporation and the presence of immature and transitional forms indicate a possible origin of all the cell types concerned from a common undifferentiated precursor. The membranes of the tubulovesicular system of FVC as well as the apical cell membrane were reactive to Thiéry's carbohydrate stain. However, lanthanum tracing of the extracellular space and ultrastructural stereoscopy did not reveal a permanent continuity between both membrane systems. The absence of 3H-thymidine label showed that FVC were not proliferative. The structural characteristics of FVC do not account for a secretory, resorptive or receptive function. The special arrangement of microfilaments and the tubulovesicular system suggests an ability to fast changes in surface area.

Animals↗
PubMed1993

Diffusion of extracellular K+ can synchronize bursting oscillations in a model islet of Langerhans.

Electrical bursting oscillations of mammalian pancreatic beta-cells are synchronous among cells within an islet. While electrical coupling among cells via gap junctions has been demonstrated, its extent and topology are unclear. The beta-cells also share an extracellular compartment in which oscillations of K+ concentration have been measured (Perez-Armendariz and Atwater, 1985). These oscillations (1-2 mM) are synchronous with the burst pattern, and apparently are caused by the oscillating voltage-dependent membrane currents: Extracellular K+ concentration (Ke) rises during the depolarized active (spiking) phase and falls during the hyperpolarized silent phase. Because raising Ke depolarizes the cell membrane by increasing the potassium reversal potential (VK), any cell in the active phase should recruit nonspiking cells into the active phase. The opposite is predicted for the silent phase. This positive feedback system might couple the cells' electrical activity and synchronize bursting. We have explored this possibility using a theoretical model for bursting of beta-cells (Sherman et al., 1988) and K+ diffusion in the extracellular space of an islet. Computer simulations demonstrate that the bursts synchronize very quickly (within one burst) without gap junctional coupling among the cells. The shape and amplitude of computed Ke oscillations resemble those seen in experiments for certain parameter ranges. The model cells synchronize with exterior cells leading, though incorporating heterogeneous cell properties can allow interior cells to lead. The model islet can also be forced to oscillate at both faster and slower frequencies using periodic pulses of higher K+ in the medium surrounding the islet. Phase plane analysis was used to understand the synchronization mechanism. The results of our model suggest that diffusion of extracellular K+ may contribute to coupling and synchronization of electrical oscillations in beta-cells within an islet.

Animals↗
PubMed1977

The site of insulin resistance after injury.

Intracellular glucose concentrations in skeletal muscle have been measured in normal and injured rats individually infused with 51Cr-EDTA as an extracellular space marker. In normal rats, even when infused with glucose at a high rate, membrane transport appeared to be the rate-limiting step in glucose utilization by muscle. After injury the concentration of intracellular glucose was increased, and positively correlated with that in plasma, showing that the loss of sensitivity to insulin after injury is due to impairment of an intracellular metabolic step.

Animals↗
PubMed1978

Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos. IV. Biochemical and autoradiographic observations on fucose-, glucose-, and mannose-labelled materials.

Normal Rana pipiens gastrulae show more incorporation of isotopically labelled fucose, glucose, and mannose into TCA-insoluble materials than blastulae. Interspecific hybrid embryos which undergo developmental arrest at the onset of gastrulation often synthesize reduced amounts of fucose-, glucose- and mannose-labelled materials. These materials are high-molecular weight and are degraded by Pronase into fragments which are included in Sepharose CL-4B but excluded from Sephadex G-50. Labelled materials contain label predominantly in fucose and galactose with little label in glucose or mannose. Labelled materials migrate slowly on cellulose acetate, bind to DEAE-cellulose and elute at low ionic strength and are precipitated by cetyl pyridinium chloride (CPC) without the addition of carrier compounds. Pulse-chase labelling experiments and light- and electron-microscopic autoradiography were used to examine the site of synthesis, mode of transport, and sites of deposition of fucose-, glucose- and mannose-labelled materials in different developmental stages of normal developing Rana pipiens embryos and interspecific hybrid embryos formed by fertilizing the eggs of R. pipiens with the sperm of R. catesbeiana. In both normal and hybrid embryos, after a 15-30-min pulse, grains are closely associated with juxtanuclear and cytoplasmic collections of membrane-bound vesicles which resemble the Golgi apparatus. In normal embryos following a 15-30-min pulse and a 60-min chase, grains are largely cleared from the cytoplasmic vesicles and deposited in the extracellular spaces or along cell surfaces. In contrast, arrested hybrid embryos given a 15-30-min pulse and a 60-min chase show a marked accumulation of grains over cytoplasmic structures such as the Golgi apparatus and vesicular elements in the cell cortex. Certain interesting features of regional variation in synthetic activity in developing normal embryos are also described.

Animals↗
PubMed1997

Modeling the route of administration-based enhancement in the brain delivery of EAB 515, studied by microdialysis.

EAB 515 (S-alpha-amino-5-phosphonomethyl[1,1'biphenyl]-3-propanoic acid) is an extremely hydrophilic N-methyl-D-aspartate antagonist. It shows marked CNS activity, in that it is a potent neuroprotector in models of cerebral ischemia, and also demonstrates social and non-social behavioral alteration following systemic administration in animals. Because of its high degree of ionization at physiologic pH, one would not expect appreciable brain uptake of EAB 515 across tight junctions of the blood-brain barrier. This is in contrast to its pharmacologic effect as well as brain/plasma ratios measured during systemic administration in rats. These observations lead us to investigate other transport pathways that might account for its brain uptake. Such mechanistic information is imperative in rational drug delivery and drug design strategies. Upon intracerebroventricular administration, the observed steady-state cortical extracellular fluid concentrations of EAB 515 were over 100-fold higher than those observed following intravenous administration, when normalized for the dosing rate. This increased distribution into the brain, based upon the route of administration, suggests the transport of drug directly between the cerebrospinal fluid and the brain extracellular space. The parameters of the model that adequately describes the data obtained from the two routes of administration in individual animals were estimated. The clinical significance of these results is in the use of intracerebroventricular administration for enhanced brain delivery of hydrophilic drugs that poorly cross the blood-brain barrier.

Animals↗