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Ultrastructure of chick tendon fibroblast with special reference to secretory mechanism.

Electron-microscope study of chick tendon fibroblast revealed a constant ultrastructural finding of a microtubular-microfibrillar system and an intimate relationship which existed between the microtubular-microfibrillar system and secretory vacuoles. Additionally, the data from this study suggest a mechanism by which newly synthesized collagen molecules are transported and secreted into the extracellular space to be organized into mature collagen.

Animals↗

Role of the extracellular Ca2+ on the intracellular Ca2+ changes in fertilized and activated mouse oocytes.

Changes in intracellular calcium concentration ([Ca2+]i) in fertilized mouse oocytes were measured using the calcium-sensitive dye, fura-2. After fertilization, an initial long-lasting [Ca2+]i increase was followed by a periodic [Ca2+]i increase. The periodic increase in [Ca2+]i persisted for 1 to 3 h and all fertilized oocytes extruded the second polar body within 4 h. The mean interval of periodic [Ca2+]i increase was 470 +/- 180 s (mean +/- SD). The frequency of the periodic [Ca2+]i increase depended on the extracellular calcium concentration. Verapamil and nifedipine inhibited the periodic [Ca2+]i increase. Sixty-five per cent of tested cells extruded the second polar body within 90 min of exposure to 7% ethanol. In these activated oocytes, the long-lasting [Ca2+]i increase was observed. However, no cells showed a repetitive increase in [Ca2+]i. Both release of calcium from intracellular stores and influx of extracellular calcium contribute to the increase in [Ca2+]i induced by ethanol. We conclude that the extrusion of the second polar body requires an increase in [Ca2+]i above a certain threshold level and the mobilization of calcium of both the intracellular and extracellular space in mouse oocytes.

Animals↗

Inhibition of glutamate uptake induces progressive accumulation of extracellular glutamate and neuronal damage in rat cortical cultures.

It is known that neurons exposed to high concentrations of glutamate degenerate and die. The clearance of this amino acid from the extracellular space depends on their active transport by Na(+)-dependent high-affinity carriers. In the present study we tested whether inhibition of glutamate transport in mixed glial/neuronal cortical cultures induces accumulation of extracellular glutamate and whether such increase results in cell damage. Three inhibitors of glutamate transport were used: L-trans-pyrrolidine-2,4-dicarboxylate (PDC), DL-threo-beta-hydroxyaspartate (THA), and dihydrokainate (DHK). Cell damage was assessed by light microscopy observations, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and leakage of lactate dehydrogenase. PDC induced a significant concentration- and time-dependent neuronal damage, whereas pure glial cultures were not affected. A good correlation was found between this damage and elevations of glutamate concentration in the medium. These effects of PDC were similar in glutamine-free medium and in medium supplemented with glutamine. THA induced identical cell damage and elevations of extracellular glutamate to those produced by PDC, while DHK did not affect at all any of these parameters. PDC- and THA-induced toxicity was protected by the N-methyl-D-aspartate receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo-(a,d)cyclohepten-5,10-imine maleate but not by the non-N-methyl-D-aspartate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline.

Animals↗

Kinetics of myo-inositol transport in rat ocular lens.

Myo-inositol (MI) influx as a function of concentration in rat lens consisted of a saturable component, fit by a rectangular hyperbola, and a linear component which was more distinct at high myo-inositol concentrations suggesting passive diffusion. The hyperbolic component was half-maximally saturated (Kt) at 61.3 microM and had a maximal transport rate (Jmax) of 44.6 mumol/kg wet wt/h. The linear component had an apparent permeability coefficient of 1.44 x 10(-6) s-1. Sorbitol, which distributed rapidly in the extracellular space (6.83 ml/100 g wet wt), also appeared to enter the intracellular space with a permeability coefficient of 1.37 x 10(-6) s-1, similar to that of myo-inositol. The influx of myo-inositol was critically dependent on the concentration of extracellular sodium consistent with a sodium-myo-inositol cotransport. The kinetics of influx activation by sodium suggested an apparent 2:1 coupling ratio for sodium and myo-inositol. When potassium was used as sodium substitute, a significantly stronger influx inhibition was observed than with nondepolarizing sodium substitutes, indicating that myo-inositol was driven by the electrochemical gradient of sodium rather than the chemical gradient only. Reducing the extracellular Na concentration increased the MI concentration at which transport was half-maximally activated, suggesting an ordered binding sequence of Na followed by MI. Myo-inositol influx was competitively inhibited by phlorizin with an inhibitory coefficient (Ki) of 35 microM. Phloretin also was capable of inhibition but with a much lesser efficacy. Myo-inositol desaturates from the lens at a rate of 0.00862 h-1. Approximately 19% of the efflux can be inhibited with phlorizin, suggesting that it represents carrier-mediated flux. The phlorizin insensitive flux has a rate of 0.00695 h-1 or 1.93 x 10(-6) s-1, similar to the Na-independent passive influx. MI influx is due to a Na-dependent, phlorizin-sensitive active transport while the efflux consists largely of a phlorizin-independent passive leakage.

Animals↗

Inhibition of membrane-bound carbonic anhydrase decreases subretinal pH and volume.

PURPOSE: The lipophilic carbonic anhydrase (CA) inhibitor acetazolamide has been shown to enhance subretinal fluid resorption, reduce subretinal pH, and can improve cystoid macular edema, but its clinical use is limited by systemic side effects. While these are most likely a result of inhibiting intracellular CA isoenzymes, retinal pigment epithelial (RPE) transport is thought to be modulated via membrane-bound CA. This study investigates whether benzolamide, a hydrophilic CA inhibitor that does not readily penetrate cell membranes, is sufficient to modulate subretinal volume and pH. METHODS: Volume and pH were assessed in the subretinal space (SRS) of the perfused chick retina-RPE-choroid preparation by calculating these variables from data obtained with two different double-barreled, ion-selective electrodes (H+ for pH and the extracellular space marker tetramethylammonium (TMA+) for SRS volume). Light induced variations and changes in baseline measurements were recorded before and after addition of 10(-4) M acetazolamide or benzolamide to the basal perfusion. RESULTS: Basal perfusion with either drug induced both an acidification of the SRS by 0.02-0.04 pH units, which occurred within 60 s, as well as an increase in the amplitude of the light-induced alkalinisation of the SRS. TMA+ concentration in the SRS increased steadily over a period of several minutes after basal perfusion with either of the CA inhibitors, and the calculated SRS volume was reduced by 40% within 8-10 min. CONCLUSION: The observation that benzolamide had effects equal to acetazolamide suggests that inhibition of membrane-bound CA at the basolateral membrane of the RPE is sufficient to decrease subretinal pH and volume. This may represent a clinically important mechanism for the resorption of sub- and intraretinal fluid.

Acetazolamide↗

Immediate response to light of rat pineal melatonin rhythm: analysis by in vivo microdialysis.

Melatonin in the extracellular space of the pineal gland was measured continuously for 4 consecutive days from single, freely moving rats by means of in vivo microdialysis. A robust circadian rhythm was observed in the pineal extracellular melatonin under both light-dark (LD) and continuous dark (DD) conditions, the patterns of which were almost identical for 4 days within individuals but varied substantially among individuals. The offset phase of melatonin rhythm was more stable than the onset phase. Light-induced phase shift of melatonin rhythm was measured in individual rats, which had been entrained to LD and subsequently released into DD. On the 1st day in DD, a 3-min light pulse of 200 lx was applied either at circadian time (CT) of 17 or 22 h (5 and 10 h after the dark onset, respectively). The light pulse rapidly suppressed the nocturnal melatonin level. The rate as well as the level of melatonin suppression was significantly greater by the pulse at CT22 than at CT17. A phase shift of the melatonin rhythm was calculated on the 2nd and 3rd days in DD. Significant phase delay shift was observed after the pulse at CT17 and advance shift after the pulse at CT22 of approximately 1 h in either case. Because the amount of phase shift was not different between the 2nd and 3rd days in DD, the phase shift of pineal melatonin rhythm by single light pulse seems to be completed immediately.

Animals↗

Ultrastructural evidence for sinusoid spaces and coupling between pituicytes in the rat.

The pericapillary palisade of the rat neurohypophysis was examined by means of thin-section and freeze-etch electron-microscopy. Special attention was given to pituicyte processes intermingled with neurosecretory terminals. These processes are identified by the presence of lipid droplets and ribosomes. Extracellular spaces are conspicuously enlarged in circumscribed regions between fingerlike protrusions of pituicyte processes. Neurosecretory axons seem to have free access to these enlarged spaces. Zonulae occludentes often combined with small gap junctions are found at the border of these sinusoid spaces. Gap junctions and occasionally intermediate junctions are seen between pituicyte processes. The topographic relationship and the functional significance of these structural features remain to be further elucidated.

Animals↗

The role of intracellular and extracellular calcium in mechanical and intracellular electrical activity of human urinary bladder smooth muscle.

We studied the role of extracellular and intracellular Ca2+ in human detrusor smooth muscle contraction. Simultaneous recordings of mechanical and intracellular electrical activity were made in three different Ca2+ concentrations: normal Krebs' solution (100%), 10% of the standard Ca2+ concentration and a solution in which Ca2+ was omitted from the medium (0%). Spontaneous contractions and KCl or CCh induced contractions were studied. Ryanodine and caffeine were used to manipulate the intracellular Ca2+ stores. The present results show that only a very small amount of Ca2+ in the extracellular space is sufficient to support spontaneous and induced contractions. Spike-shaped potentials and long lasting depolarisations were recorded in all three solutions. However, the prevalence of long lasting depolarisations increased when the extracellular Ca2+ concentration was reduced. The amplitude of the spike-shaped potentials and long lasting depolarisations appeared to be negatively affected by diminishing the extracellular Ca2+ concentration. Additionally, the duration of the long lasting depolarisations was reduced in 0% Ca2+. The contraction upon KCl stimulation was primarily depending on the extracellular Ca2+. Upon muscarinic receptor stimulation, a combined activation of Ca2+ mobilisation from intracellular and extracellular stores may occur; the ratio of contribution of these two sources changes in accordance with the requirements of the conditions.

Acetylcholine↗

THE FINE STRUCTURE OF THE ELECTRIC ORGAN OF TORPEDO MARMORATA.

The fine structure of the electric organ of the fish Torpedo marmorata has been examined after osmium tetroxide or potassium permanganate fixation, acetone dehydration, and Araldite embedment. This organ consists of stacks of electroplaques which possess a dorsal noninnervated and a ventral richly innervated surface. Both surfaces are covered with a thin basement membrane. A tubular membranous network whose lumen is continuous with the extracellular space occupies the dorsal third of the electroplaque. Nerve endings, separated from the ventral surface of the electroplaque by a thin basement membrane, contain synaptic vesicles (diameter 300 to 1200 A), mitochondria, and electron-opaque granules (diameter 300 A). Projections from the nerve endings occupy the lumina of the finger-like invaginations of the ventral surface. The cytoplasm of the electroplaques contains the usual organelles. A "cellular cuff" surrounds most of the nerve fibers in the intercellular space, and is separated from the nerve fibre and its Schwann cell by a space containing connective tissue fibrils. The connective tissue fibrils and fibroblasts in the intercellular space are primarily associated with the dorsal surface of the electroplaque.

Animals↗

Structure and development of viruses as observed in the electron microscope. VI. ECHO virus, type 9.

Sequential stages in the development and release of ECHO 9 virus have been illustrated and described. It is suggested that viral particles differentiate and become oriented in columns upon a fine filamentous lattice at cytoplasmic template sites which are distinct from the endoplasmic reticulum. Subsequently, virus is dispersed in the peripheral cytoplasm and gains egress from the cell through rents in the plasma membrane. Complete cellular disruption with viral release may supervene. The virus consists of a 13 to 15 mmicro dense core and a poorly defined outer membrane, 22 to 24 mmicro in diameter. Incomplete forms, lacking the core, are observed in the cytoplasm but have not been seen in the extracellular space.

Animals↗

Taurine release evoked by NMDA receptor activation is largely dependent on calcium mobilization from intracellular stores.

It is known that the activation of N-methyl-D-aspartate (NMDA) receptors leads to an increase in extracellular taurine concentration in different brain regions. The mechanism that mediates this effect is not totally understood. In this study, rat hippocampal slices were used to determine the dependence of NMDA-induced taurine release on extracellular calcium and/or on calcium mobilization from intracellular stores. NMDA was administered through a microdialysis probe inserted into the slice, at the level of CA1 stratum radiatum, which was also used to collect amino acids from the extracellular space. Field potentials evoked by stimulation of the Schaffer collaterals and recorded in the stratum pyramidale of CA1 were used as a control of NMDA receptor activation. NMDA induced a marked increase in extracellular taurine levels and a decrease in field potential amplitude, and both effects were suppressed in the presence of MK-801, a blocker of the NMDA receptor-linked channel. Dantrolene, an inhibitor of calcium release from intracellular stores, partially inhibited the extracellular taurine increase, while 2-nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phosphatidylinositol-specific phospholipase C activation, had no effect. Removal of extracellular calcium diminished, but did not abolish, the extracellular taurine increase caused by NMDA. The remaining taurine response was totally suppressed by dantrolene, and also by NCDC. These results demonstrate that the release of taurine induced by NMDA receptor activation is triggered by the increase in cytoplasmic calcium concentration. We suggest that, under physiological conditions, calcium influx provides the signal for NMDA-induced taurine release, which is amplified by calcium-dependent calcium mobilization from intracellular stores.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Correlations between the apparent diffusion coefficient, water content, and ultrastructure after induction of vasogenic brain edema in cats.

OBJECT: The authors examined the correlation between changes in the apparent diffusion coefficient, regional water content, and tissue ultrastructure after vasogenic brain edema. METHODS: Vasogenic edema was induced in the white matter of six cats by cortical cold lesioning. The trace of diffusion tensor (Trace[D]) obtained from magnetic resonance imaging to measure the orientationally averaged water diffusibility was compared with the corresponding tissue water content determined by gravimetric studies and with ultrastructural water localization. Edema fluid had spread to the subcortical and deep white matter by 4.5 hours postlesioning. The increase in Trace(D) showed a significant linear correlation with the increase in tissue water content, both in the subcortical and deep white matter as follows: y = 45.5x - 2367 (r = 0.94) and y = 37.0x - 1769 (r = 0.93), respectively, where x is the water content (gram water/gram tissue) and y the Trace(D) (x 10(-6) mm2/second). On histological examination, nerve fibers were found to be dissociated in the white matter and the extracellular space was markedly enlarged with protein-rich fluid. No noticeable hydropic swelling of the cellular components was observed. CONCLUSIONS: A linear correlation was observed between increases in Trace(D) and increases in extracellular water volume in in vivo vasogenic brain edema. A similar correlation between the subcortical and deep white matter showing different arrangements of nerve fibers (parallel compared with intermingled, respectively) indicated that measurement of Trace(D) is a suitable parameter for the evaluation of vasogenic brain edema.

Animals↗

[Measuring electric impedance of organs--methodologic principles].

Ischemia causes changes in organ tissue (e.g. during operation or transplantation) which may finally lead to irreversible injury, so that the organ can no longer be resuscitated. To the extent that these changes affect the electrical properties of the tissue they are manifested in the impedance spectrum. As an example, the course of impedance of a HTK-protected porcine liver is presented in the frequency range of 0.1 Hz to 10 MHz, which includes two dispersion--alpha- and beta-dispersion. Using a suitable electrical equivalent circuit analogue to the structure of the liver, the behavior of the alpha- and beta-dispersion is explained on the basis of gap junction closure and narrowing of the extracellular space due to cell swelling.

Animals↗

A microstructural study of spinal cord edema.

The experimental spinal cord edema was produced in a cat by the infusion method of Marmarou. Horseradish peroxidase (HRP) dissolved in autoserum of a cat was used as a tracer. After laminectomy, a 30-gauge needle was inserted into the intumescentia cervicalis. A total amount of 20 microliters of a tracer was infused at a rate of 10 microliters/hr. The structural features were studied immediately and 3 days after infusion. Immediately after infusion, HRP was noted in the infused white and gray matters. Though the perivascular space in the white matter at the infused site was widely distended and filled with HRP, the space in the gray matter was not distended but filled with HRP. HRP which was observed along vessels led to the surface of the spinal cord. Swelling of astrocyte was not observed. Three days after infusion, the extracellular space and the perivascular space in the infused white matter were still expanded but were not filled with HRP. The fine structural features were similar to the findings as seen in Marmarou's infusion type of brain edema. Using this model, it seems to be feasible to study the resolution process of spinal cord edema.

Animals↗

Perilymphatic fluid compartments and intercellular spaces of the inner ear and the organ of Corti.

An in vitro preparation of the inner ear cochlea has been used to visualize the structural relationships of unfixed, living sensory cells and structural components within the intact hearing organ. By perfusing perilymphatic compartments of the cochlea with fluorochrome-conjugated dextran, the extracellular spaces were clearly outlined. The staining pattern illustrated the large fluid compartments formed by the tunnel of Corti, the space of Nuel, and the outer tunnel. The dextran solution also indicated the spaces between the outer hair cell rows, the inner hair cells, and the surrounding supporting cells. The staining pattern demonstrates that the organ of Corti has a loose structure, suggesting a weak mechanical coupling between the cells. Moreover, it is evident that substances applied to the perilymph (e.g., therapeutic drugs) will readily reach all the cells of the hearing organ. In addition to the intraorgan fluid compartments, the spiral limbus was shown to contain significant volumes of perilymph within the intercellular spaces forming the so-called teeth of Huschke between the interdental cells. An extensive system of bundles following the teeth of Huschke was shown to be completely immersed in perilymph. The bundles were stained by a potentiometric dye, which in the inner ear primarily stains nerve fibers and sensory cells, which may indicate a nervous control of cells in this region.

Animals↗

Stimulus- and amino acid-induced calcium and potassium changes in rat neocortex.

Local changes in extracellular concentration of calcium ions, [Ca2+]o, and potassium ions, [K+]o, elicited through ionophoretic applications of the excitatory amino acids glutamate (Glu) and aspartate (Asp) or through cortical surface stimulations were measured in the rat motor cortex using paired ion-selective microelectrodes. Glu or Asp applications produced dose-dependent decreases in [Ca2+]o from a base line of 1.25 mM to as low as 0.08 mM and increases in [K+]o from 2.8 mM to as high as 13.3 mM. These ionic changes were accompanied by negative DC potential shifts of up to 15 mV. [K+]o changes were practically constant in all cortical layers, whereas [Ca2+]o changes were variable with depth, showing two localized marked maximums. One was observed in the superficial layers, 150-300 microns below cortical surface and the second at 1,100-1,300 microns, in a region corresponding roughly to layer V. Application of tetrodotoxin (TTX) onto the cortical surface abolished stimulus-evoked ionic changes, whereas amino acid-induced [Ca2+]o changes remained practically unaltered, and [K+]o changes were reduced by 20% on average. Selective degeneration of the pyramidal tract neurons was induced by performing chronic lesions of the pyramidal tract at the bulbar level. In these conditions, laminar profiles of amino acid-induced [Ca2+]o changes were considerably altered in the corresponding motor cortex. The maximum Ca2+ changes were then reduced by two-thirds, and the [Ca2+]o signals were practically constant throughout the cortex. Local cobalt (Co2+) or manganese (Mn2+) applications could abolish amino acid-evoked [Ca2+]o changes, whereas most of the [K+]o changes persisted. gamma-Aminobutyric acid applications could decrease [Ca2+]o signals to a large extent, whereas [K+]o changes were diminished by only 15-25% on average. These results show that the putative neurotransmitters Glu and Asp induce significant changes in [Ca2+]o and [K+]o in the rat cerebral cortex and indicate the possible origin of the ion fluxes in terms of neuronal elements and ionic channels: in particular, they indicate that the major part of the amino acid-induced [Ca2+]o maximum decreases can be ascribed to Ca2+ movements through channels located very likely on the pyramidal tract neurons in the motor cortex. They also indicate that significant reductions of the extracellular space volume occur during excitatory amino acid applications.

Amino Acids↗

Long-spacing collagen in skin biopsies from patients with lepromatous leprosy.

In the course fo evaluating the effect of different drugs on the morphology of Mycobacterium leprae (Edwards, Draper & Draper, 1972) and on the phagocytic cells containing these organisms (Edwards, 1973), an unusual feature was observed in the dermal connective tissue of skin biopsies obtained from some of the patients. Variable amounts of an extracellular, cross-striated osmiophilic material were present in the dermis. The purpose of this communication is to describe the banded substance observed in the extracellular space of the lepromatous skin and to compare it briefly with similar substances and structures reported to occur elsewhere.

Animals↗

Cellular mechanisms of normal growth in the mammalian heart. I. Qualitative and quantitative features of ventricular architecture in the dog from birth to five months of age.

This paper describes the qualitative and quantitative composition of dog myocardium over the first 5 months of life. The quantitative composition of dog right and left ventricle over this period does not vary. A stereological analysis of electron micrographs representing 32,000 micron2 of tissue surface revealed that 79% of the heart is made up of myofibers, whereas 21% is extracellular space. Twenty-eight percent of the extracellular compartment by volume is vasculature (tissue was preserved by immersion rather than vascular perfusion); 72% is occupied by nonvascular elements and "empty" space. In contrast to the remarkable constancy of quantitative composition of the whole myocardium, myocyte shape and dimensions and the arrangement of intercellular connections vary dramatically over the age period studied. In early postnatal life, the morphology of blood vessels, many of which have completely partitioned lumina, also changes significantly.

Aging↗