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PubMed1999

Barrier mechanisms in the brain, II. Immature brain.

1. It is widely believed that 'the' blood-brain barrier is immature in foetuses and newborns. 2. Much evidence in support of this belief is based on experiments that were unphysiological and likely to have disrupted fragile blood vessels of the developing brain. Some confusion about barrier development arises from insufficient recognition that the term 'blood-brain barrier' describes a complex series of mechanisms controlling the internal environment of the brain. 3. We present evidence showing that the brain develops within an environment that, particularly with respect to protein, is different from that of the rest of the body and that possesses a number of unique features not present in the adult. 4. Barriers to protein at blood-brain and blood-cerebrospinal fluid (CSF) interfaces (tight junctions) are present from very early in development; immunocytochemical and permeability data show that proteins are largely excluded from extracellular space in developing brain. 5. Cerebrospinal fluid in developing brain contains high concentrations of proteins largely derived from plasma. This protein is transferred from blood by an intracellular mechanism across the epithelial cells of the immature choroid plexus. Only a small proportion of choroid plexus cells is involved. The route is an intracellular system of tubulo-endoplasmic reticulum continuously connected across the epithelial cells only early in brain development. 6. High concentrations of proteins in CSF in developing brain are largely excluded from the brain's extracellular space by barriers at the internal and external CSF-brain interfaces. These consist of membrane specializations between surfaces of cells forming these interfaces (neuroependyma on the inner surface; radial glial end feet on the outer surface). In contrast with tight junctions present at the blood-brain and blood-CSF barriers, at the CSF-brain barriers of the immature brain, other junctional types are involved: strap junctions in the neuroependyma and a mixture of junctions at the outer CSF-brain barrier (plate junctions, strap junctions and wafer junctions). These barriers are not present in the adult. 7. Permeability to small lipid-insoluble molecules is greater in developing brain; more specific mechanisms, such as those involved in transfer of ions and amino acids, develop sequentially as the brain grows.

Adult↗
PubMed1989

Histamine concentration and Ca2+ mobilization in arterial smooth muscle.

Smooth muscle contraction is dependent on Ca2+ entry from the extracellular space or release from intracellular stores. The sensitivity of these Ca2+ sources to agonist concentration was evaluated by measuring myoplasmic [Ca2+] (as estimated by aequorin), myosin phosphorylation, and isometric stress in the swine carotid media. High histamine concentrations produced transient elevations in [Ca2+] and phosphorylation with rapid generation of near maximal stress. Lower histamine concentrations produced much smaller [Ca2+] and phosphorylation transients, and stress development was slower. Peak [Ca2+] was proportional to the rate of stress development. Steady-state [Ca2+], phosphorylation, and stress values (which are dependent on extracellular Ca2+) were more sensitive to histamine concentration than was the peak [Ca2+] response both in the presence and absence of extracellular CaCl2 (measures of intracellular Ca2+ release). This result suggests that the mechanism for Ca2+ influx from the extracellular space is more sensitive to histamine than intracellular Ca2+ release. These results are also consistent with the hypothesis that agonist-releasable sarcoplasmic reticular Ca2+ is the major contributor to initial phosphorylation transients that enhance the rate of stress development.

Animals↗
PubMed1989

Chloride enters glial cells and photoreceptors in response to light stimulation in the retina of the honey bee drone.

Double-barrelled ion-selective microelectrodes were used to measure free [Cl-] in photoreceptors, extracellular space, and glial cells in superfused slices of drone retina. Tests indicated that with normal superfusate the intracellular electrode signal was due essentially to Cl- and not to some other interfering anion. The results indicate that Cl- is more concentrated in both photoreceptors and glial cells than would be predicted for a passive electrochemical distribution. When the photoreceptors were stimulated by a standard train of 20 ms flashes, 1/s for 90 s, their intracellular free [Cl-] (Cli) rose by 8 +/- 1 mM. At the end of stimulation Cli usually continued to rise for up to a further 2 min and then returned toward the baseline over about 10 min. During light stimulation Cli in the glia rose. The magnitude of the increase was 5.1 +/- 0.4 mM, about half the increase in Ki. In some extracellular recording sites, light stimulation caused [Cl-] to increase and in others to decrease. The mean change was -0.7 mM, SD 6.5 mM. The Cl- that entered the photoreceptors and the glia was presumably made available by the shrinking of the extracellular space. When the cells were depolarized by increasing [K+] in the superfusate from 7.5 mM to 18 mM, Cli increased. The half-time of the change in Cli was longer than the half-time of the depolarization by 10-30 s in the glia and 50-250s in the photoreceptors. During superfusion with 0 Cl- Ringer's solution, the light-induced rise in extracellular [K+] was greater by a factor of 1.4-2.7, and the clearance after the end of the stimulation was slower. The rate of increase in glial Ki during light stimulation fell; the rate of increase of glial Ki caused by superfusion with raised [K+] (in the absence of Cl-) fell more. We conclude that when extracellular [K+] is increased, entry of Cl- into the glia is necessary for part, but not all, of the net uptake of K+. During light stimulation, the observed movement of CL- into glia contributes to homeostasis of extracellular [K+], and the cell swelling associated with movement of Cl- into both glia and photoreceptors contributes to homeostasis of extracellular [Na+].

Animals↗
PubMed2001

Effects of chemotherapy by 1,3-bis(2-chloroethyl)-1-nitrosourea on single-quantum- and triple-quantum-filtered 23Na and 31P nuclear magnetic resonance of the subcutaneously implanted 9L glioma.

The effects of chemotherapy [25 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea administered with a single i.p. injection] on cellular energetics by 31P nuclear magnetic resonance (NMR) spectroscopy, total tissue sodium by single-quantum (SQ) 23Na NMR spectroscopy, and intracellular sodium by triple-quantum-filtered (TQF) 23Na NMR spectroscopy were studied in the s.c. 9L glioma. Animals were studied by NMR 2 days before therapy and 1 and 5 days after therapy. Destructive chemical analysis was also performed 5 days after therapy to validate the origin of changes in SQ and TQF 23Na signals. One day after treatment, there was no significant difference between control and treated tumors in terms of tumor size or 23Na and 31P spectral data. Five days after therapy, treated tumors had 28 +/- 16% (P < 0.1) lower SQ 23Na signal intensity, 46 +/- 20% (P < 0.05) lower TQF 23Na signal intensity, 125 +/- 51% (P < 0.05) higher ATP:Pi ratio, 186 +/- 69% (P < 0.05) higher phosphocreatine:Pi ratio, and 0.17 +/- 0.06 pH units (P < 0.05) higher intracellular pH compared with control tumors. No significant differences in TQF 23Na relaxation times were seen between control and treated tumors at any time point. Destructive chemical analysis showed that the relative extracellular space of control and treated tumors was identical, but the treated tumors had 21 +/- 8% (P < 0.05) lower total tissue Na+ concentration and 60 +/- 24% (P < 0.05) lower intracellular Na+ concentration compared with the controls. The higher phosphocreatine:Pi and ATP:Pi ratios after 1,3-bis(2-chloroethyl)-1-nitrosourea treatment indicate improved bioenergetic status in the surviving tumor cells. The decrease in SQ and multiple-quantum-filtered 23Na signal intensity was largely attributable to a decrease in Na(i)+ because the treatment did not change the relative extracellular space. The improved energy metabolism could decrease the intracellular concentration of Na+ by increasing the activity of Na+-K+-ATPase and decreasing the activity of Na+/H+. Although both 23Na and 31P spectra were consistent with improved cellular metabolism in treated tumors, the 23Na methods may be better suited for monitoring response to therapy because of higher signal:noise ratio and ease of imaging the single 23Na resonance.

Adenosine Triphosphate↗
PubMed1997

Hypothyroidism delays fetal stratum corneum development in mice.

The epidermal permeability barrier, required for terrestrial life, is localized to lipid-enriched lamellar membranes in the extracellular spaces of the stratum corneum (SC). Immaturity of the SC is a significant contributor to morbidity and mortality in premature infants. Previous studies have shown that supraphysiologic concentrations of thyroid hormone accelerate epidermis/SC ontogenesis. Here we studied SC development in Hyt/Hyt mice who are genetically hypothyroid due to a mutation in the TSH receptor. In control mice on d 18 of gestation (term 19.5 d), only focal areas displayed a mature SC membrane pattern. By 19 d of gestation there was a mature multilayered SC with lamellar unit structures filling the extracellular spaces similar to that seen in mature mice. In Hyt/Hyt mice SC development was delayed at both 18 and 19 d of gestation. In both strains of mice, within the first day after birth there were no differences in epidermal or SC appearance, and the SC was fully mature. These findings indicate that thyroid hormone plays a physiologic role during normal intrauterine development of the SC. However, normal SC maturation ultimately occurs, indicating that thyroid hormone is not absolutely essential. Previous studies have shown that glucocorticoids accelerate SC development in euthyroid rats, and in the present study we demonstrate that glucocorticoids also accelerate SC ontogenesis in euthyroid mice. In contrast, in Hyt/Hyt mice glucocorticoids did not accelerate or normalize SC development, indicating that the glucocorticoid effect on SC maturation requires a euthyroid state or that glucocorticoids act via thyroid hormone. These studies demonstrate that thyroid hormone status is an important regulator of fetal SC development.

Animals↗
PubMed1991

Aluminum distribution into brain and liver of rats and rabbits following intravenous aluminum lactate or citrate: a microdialysis study.

Microdialysis probes were utilized to follow the appearance and disappearance of dialyzable aluminum (Al) in rat and rabbit brain and liver extracellular fluid compared to blood after iv Al lactate or Al citrate injection. Dialyzable Al was assumed to be the fraction not protein bound or self-associated into complexes greater than the molecular weight cutoff of the dialysis membrane. Aluminum concentrations peaked in brain frontal cortex and ventral hippocampus and in the liver in the first 20-min dialysis sample, indicating rapid Al penetration into the extracellular space of these organs. In vitro recovery experiments conducted with microdialysis probes at room temperature revealed an average dialysis efficiency of about 10% for both Al lactate and citrate. At 37 degrees C Al recovery increased for both Al lactate and citrate. In vivo Al recovery from rabbit blood averaged 5.15% for Al lactate and 3.25% for Al citrate. These observations are consistent with results from recovery studies of other substances showing an increased recovery with increased temperature but an overestimate of recovery by in vitro methods. Tissue/blood Al ratios (TBR; representing dialyzable extracellular tissue Al divided by dialyzable blood plasma Al) for liver were approximately 1, suggesting unhindered diffusion of Al between blood and liver. In contrast, brain TBR were less than 1, demonstrating a partial blood-brain barrier to Al. The brain TBR for Al lactate was greater than TBR for Al citrate, suggesting that Al citrate did not preferentially penetrate the blood-brain barrier. Higher TBR were seen in the rabbit than the rat, perhaps contributing to the greater susceptibility of the rabbit to Al-induced neurobehavioral toxicity. Metals can be repetitively sampled in the extracellular space using microdialysis, enabling metal toxicokinetic determinations in these compartments.

Aluminum↗
PubMed2004

Regulation of bone morphogenetic proteins in early embryonic development.

Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-beta family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral - or back to belly - body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd-BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

Animals↗
PubMed1979

Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport.

The fate of tetanus toxin (mol wt 150,000) subsequent to its retrograde axonal transport in peripheral sympathetic neurons of the rat was studied by both electron microscope autoradiography and cytochemistry using toxin-horseradish peroxidase (HRP) coupling products, and compared to that of nerve growth factor (NGF), cholera toxin, and the lectins wheat germ agglutinin (WGA), phytohaemagglutinin (PHA), and ricin. All these macromolecules are taken up by adrenergic nerve terminals and transported retrogradely in a selective, highly efficient manner. This selective uptake and transport is a consequence of the binding of these macromolecules to specific receptive sites on the nerve terminal membrane. All these ligands are transported in the axons within smooth vesicles, cisternae, and tubules. In the cell bodies these membrane compartments fuse and most of the transported macromolecules are finally incorporated into lysosomes. The cell nuclei, the parallel golgi cisternae, and the extracellular space always remain unlabeled. In case the tetanus toxin, however, a substantial fraction of the labeled material appears in presynaptic cholinergic nerve terminals which innervate the labeled ganglion cells. In these terminals tetanus toxin-HRP is localized in 500-1,000 A diam vesicles. In contrast, such a retrograde transsynaptic transfer is not at all or only very rarely detectable after retrograde transport of cholera toxin, NGF, WGA, PHA, or ricin. An atoxic fragment of the tetanus toxin, which contains the ganglioside-binding site, behaves like intact toxin. With all these macromolecules, the extracellular space and the glial cells in the ganglion remain unlabeled. We conclude that the selectivity of this transsynaptic transfer of tetanus toxin is due to a selective release of the toxin from the postsynaptic dendrites. This release is immediately followed by an uptake into the presynaptic terminals.

Adrenergic Fibers↗
PubMed2002

The role of inhibitory amino acidergic neurotransmission in hepatic encephalopathy: a critical overview.

Gamma-Aminobutyric acid (GABA) is the main inhibitory amino acid in the central nervous system (CNS). Experiments with animal models of HE, and with brain slices or cultured CNS cells treated with ammonia, have documented changes in GABA distribution and transport, and modulation of the responses of both the GABA(A)-benzodiazepine receptor complex and GABA(B) receptors. Although many of the data point to an enhancement of GABAergic transmission probably contributing to HE, the evidence is not unequivocal. The major weaknesses of the GABA theory are (1) in a vast majority of HE models, there were no alterations of GABA content in the brain tissue and/or extracellular space, indicating that exposure of neurons to GABA may not have been altered, (2) changes in the affinity and capacity of GABA receptor binding were either absent or qualitatively different in HE models of comparable severity and duration, and (3) no sound changes in the GABAergic system parameters were noted in clinical cases of HE. Taurine (Tau) is an amino acid that is thought to mimic GABA function because of its agonistic properties towards GABA(A) receptors, and to contribute to neuroprotection and osmoregulation. These effects require Tau redistribution between the different cell compartments and the extracellular space. Acute treatment with ammonia evokes massive release of radiolabeled or endogenous Tau from CNS tissues in vivo and in vitro, and the underlying mechanism of Tau release differs from the release evoked by depolarizing conditions or hypoosmotic treatment. Subacute or chronic HE, and also long-term treatment of cultured CNS cells in vitro with ammonia, increase spontaneous Tau "leakage" from the tissue. This is accompanied by a decreased potassium- or hypoosmolarity-induced release of Tau and often by cell swelling, indicating impaired osmoregulation. In in vivo models of HE, Tau leakage is manifested by its increased accumulation in the extrasynaptic space, which may promote inhibitory neurotransmission and/or cell membrane protection. In chronic HE in humans, decreased Tau content in CNS is thought to be one of the causes of cerebral edema. However, understanding of the impact of the changes in Tau content and transport on the pathogenic mechanisms of HE is hampered by the lack of clear-cut evidence regarding the various roles of Tau in the normal CNS.

Amino Acids↗
PubMed1987

Tracer permeability of rat cortical blood vessels during regional hypothermia.

Using lanthanum nitrate as a tracer, the permeability of rat cortical blood vessels was investigated during regional hypothermia of the cerebral cortex by electron microscopy. The concentration of K+ ions in the extracellular space of the cortex was determined using an ion-selective micro-electrode. Only at temperatures below about 7 degrees C was an extravasation of tracer observed in a number of cortical capillaries and arterioles, where some of the tight junctions became widened and permeable. In a few cases penetration of the tracer into vacuoles of phagocytosing pericytes or macrophages was found. The extravasation of the tracer could mainly be observed in vessels on and below the cortical surface, less often in deeper cortical parts. At the same degree of hypothermia, the K+ concentration in the extracellular space increased, reaching a maximal value of ca. 6 mmol/l at a depth of about 200 micron. As could be shown in one previous experiment, the K+ concentration reached normal values again after rewarming to 37 degrees C for 15 min, but an extravasation of the tracer was still observed. The role of the endothelial tight junctions and of the astrocytes together with the pericytes (or macrophages) is discussed regarding their importance for the effectiveness of the blood-brain barrier system.

Animals↗
PubMed1992

Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice.

Using light and electron microscopy, we investigated the in vivo distribution of liposomes sterically stabilized by specific lipids which prolong their circulation in blood. Tissue distribution of sterically stabilized liposomes composed of distearoyl phosphatidylcholine:cholesterol:monosialoganglioside GM1 (10:5:1)-encapsulated 67Ga-Desferal indicates that more than 30% of liposomes still remain in the blood at 24 h after tail vein injection. Moreover, such liposomes accumulated in tumors (C-26 colon carcinoma cells implanted s.c.), reaching almost the same level of uptake as liver (approximately 20% injected dose/g tissue). The microscopic localization of liposomes labeled with encapsulated colloidal gold or rhodamine-labeled dextran coincided well with the tissue distribution. To evaluate circulation parameters, two sizes of gold-containing egg phosphatidylcholine:cholesterol:distearoyl phosphatidylethanolamine (derivatized at its amino position with a 1900 molecular weight segment of polyethylene glycol) (10:5:0.8) liposomes were injected. The plasma was examined by electron microscopy of negative-stained preparations at 0.5, 4, and 24 h after liposome injection. It was found that the ratio of small (less than 100 nm diameter) to large (greater than 100 nm) liposomes increased with time, indicating a much faster clearance of the larger liposomes. To detect the localization of liposomes in various tissues, appropriate samples were fixed 24 h after the injection of gold-containing liposomes (between 80 and 100 nm in diameter) composed of egg phosphatidylcholine:cholesterol:monosialoganglioside GM1 (10:5:1) or egg phosphatidylcholine:cholesterol:derivatized distearoyl phosphatidylethanolamine. The tissues examined for this study included normal liver, bone marrow, and implanted neoplasms. Silver-enhanced colloidal gold was found predominantly within Kupffer cells in the normal liver and within macrophages in the bone marrow. Rarely were any silver-enhanced gold particles detected in hepatocytes. In all preparations, electron microscopy revealed the presence of gold in endosomes and lysosomes of fixed sinusoidal lining macrophages in the liver and bone marrow. Peripheral to the implanted tumors, silver enhancement revealed gold in small blood vessels and focally beyond the vessel boundaries in extracellular spaces around tumor cells. Gold particles were not observed within the tumor cell cytoplasm. At the tumor border, nonenhanced gold was occasionally seen by electron microscopy in cells of the mononuclear phagocyte system. We obtained the same localization pattern as with silver enhancement by using an alternative aqueous content marker, rhodamine B isothiocyanate-dextran. We conclude that liposomes of specific composition, which have the ability to remain in circulation with a half-life of 12-24 h, are also able to transverse the endothelium of small blood vessels, including those in tumors, and extravasate into extracellular spaces.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

Microelectrode studies of the tegument and sub-tegumental compartments of male Schistosoma mansoni: anatomical location of sources of electrical potentials.

Histological studies using horse-radish peroxidase (HRP) as a marker, injected iontophoretically through a recording electrode, have indicated the origins of potentials encountered upon advancement of the electrode into and beneath the dorsal surface of adult male Schistosoma mansoni. The first potential encountered, having a value of -51 +/- 0.6 mV originates across the outer tegumental membrane. The next potential has a value of -28 +/- 0.6 mV and originates in the muscle masses underlying the tegument. Finally, a potential having the value -10 +/- 0.5 mV originates within the basal lamina and the interstitial fibers and extracellular space surrounding the muscle. Altering ion concentrations in the bathing medium (i.e. high K+, low Na+, zero Ca2+, low Cl-, high Li+) depolarizes all three potentials. External applications of ouabain and the antischistosomal, praziquantel, also cause depolarization of the potentials. It appears that the muscle potential and the tegumental potential are primarily K+-dependent. The depolarizing effects of ouabain. LiCl and low Na+ suggest that active transport is important in the maintenance of the muscle potential, just as is the case for the tegumental potential. There appears to be a close correlation between changes in the tegumental, muscle and extracellular space potentials. The correlation between tegument and muscle potential changes might be explained by junctional complexes between tegumental cell bodies and muscle cell bodies.

Animals↗
PubMed1983

Glial endocytosis of protein in the traumatized brain.

The effect of trauma on the clearance of exogenous protein from the extracellular space of the brain was examined in monkeys and rats. Manual contusion of the monkey temporal lobe or air puff contusion of the rat parietal cortex resulted in intense glial uptake of the protein marker, horseradish peroxidase (HRP). There was no evidence of punctate or diffuse material in neurons at or distant from the trauma site. Cellular uptake was evident as early as 4 hr post trauma and reached maximal expression 24 hr post injury. By 72 hr post trauma few labeled cells could be seen. The extent of the glial uptake of protein was suggested by the decrease in extracellular marker in the vicinity of the labeled cells and by calculation of the fraction of glia so labeled. Sites of labeled glia were highly localized, corresponding to regions of stress. The corpus callosum was most clearly labeled in the rat experiments. The data suggest that in cases of traumatic injury to the brain, glia may be selectively stimulated to pronounced uptake of protein marker and thus may represent a potential means of clearing the extracellular space of extravasated serum protein.

Animals↗
PubMed2005

Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures.

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen-glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen-glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen-glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen-glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen-glucose deprivation induced necrotic cell death only. Addition of 10 microM of the N-methyl-D-aspartate glutamate receptor antagonist MK-801, and 20 microM of the non-N-methyl-D-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline to the culture medium confirmed that both N-methyl-D-aspartate and non-N-methyl-D-aspartate ionotropic glutamate receptors were involved in the oxygen-glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the DL-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of DL-threo-beta-benzyloxyaspartate in concentrations of 25 microM or more to otherwise untreated hippocampal slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen-glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10 microM) dose of DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation (but not during the next 48 h after oxygen-glucose deprivation) significantly reduced the oxygen-glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by DL-threo-beta-benzyloxyaspartate during oxygen-glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen-glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen-glucose deprivation-induced necrotic cell death in hippocampal slice cultures, which have proven to be a feasible tool in experimental studies on this topic.

Amino Acid Transport System X-AG↗
PubMed2004

Immunohistochemical detection of haem oxygenase in AA amyloidogenesis.

Haem oxygenase (HO)-1, a rate-limiting enzyme in the degradation of haem, is increased in Alzheimer's disease and in inflammations such as AA amyloidosis. However, the specific association of HO-1 is poorly understood in AA amyloidosis. In this study, we designed the experiment to reveal the contribution and association of HO-1 in the spleen during experimental murine AA amyloidosis. Experimental murine AA amyloidosis was induced with injection of an emulsion consisting of Freund's complete adjuvant and Mycobacterium butyricum. The serum amyloid A level was highest on day 3. The distribution of cells containing iron, indicating an increase of HO-1 in the red pulp, was detected with Berlin blue staining. AA amyloid formation was immunohistochemically detected as a marker by chondroitin sulphate proteoglycan (CSPG), one of the components of AA amyloid fibrils. Immunolocalizations of HO-1 and CSPG indicated a conspicuous increase and scattering of positive cells in the red pulp of the spleen. Double positive cells were not detected. On day 7, amyloid deposition was detected with Congo red staining in the extracellular spaces in the marginal zone of the white pulp in the spleen and HO-1-positive cells accumulated near the amyloid deposition area. CSPG was detected within the cells and also localized in the amyloid deposition area. CSPG was still not localized in the HO-1-positive cells. Double positive cells of HO-1 and CSPG were localized in the red pulp and in the amyloid deposition area on day 14. X-ray microanalysis indicated the existence of iron in the electron-dense bodies of fibroblasts in the amyloid deposition areas. The fibroblasts extended amyloid fibrils into the extracellular spaces of the marginal zone. These results suggest that HO-1-positive fibroblasts, but not HO-1-positive macrophages, are associated with the late stage of amyloid fibril formation.

Amyloidosis↗
PubMed1988

Electron-microscopic immunocytochemistry of 5-hydroxytryptamine in the ascidian endostyle.

The cellular and subcellular distribution of serotonin (5-hydroxytryptamine, 5-HT) in the endostyle of three species of ascidians, Ciona intestinalis, Corella parallelogramma, Ascidia mentula, was studied by light- (immunoperoxidase) and electron-microscopic (immunogold) immunocytochemistry. At the light-microscopic level 5-HT-like immunoreactivity (5-HT-LI) was exclusively found in cells located in the lateral portion of the endostyle, between zone 7, known to have iodinating capacity, and zone 8, which consists of ciliated cells. At the electron-microscopic level, the 5-HT-immunoreactive cells were found to correspond to cells containing polymorphous, dense granules, 100-300 nm in diameter. The granules were located in the supranuclear cytoplasm facing the endostyle lumen as well as in the infranuclear cytoplasm facing the extracellular space. Quantification showed that the 5-HT-LI was considerably higher (13-67 times) in cytoplasmic areas containing granules as compared to areas devoid of granules. Most, but not all, of the 5-HT-LI was associated with the dense core of the granules. In conclusion, serotonin-containing cells are located in the peripheral portion of the endostyle, between zones 7 and 8. Serotonin is stored in cytoplasmic granules that are present both in the apical and basal cytoplasm. This suggests the possibility that the cells are bipolar and secrete serotonin both in a basal direction to the extracellular space, and in an apical direction to the pharyngeal lumen.

Animals↗
PubMed1994

Changes in serotonin level in the hypoglossal nucleus region during carbachol-induced atonia.

The excitability of hypoglossal (XII) motoneurons innervating genioglossal muscles is markedly suppressed during the rapid-eye-movement (REM) stage of sleep. This may contribute to airway obstructions in sleep apnea patients. Based on our earlier studies in decerebrate cats using injections of carbachol into the pons to induce a REM sleep-like atonia and microinjections of serotonin (5HT) into the XII motor nucleus, we hypothesized that a sleep-related withdrawal of the serotonergic excitatory input to XII motoneurons may play a major role in these processes. To test one aspect of this hypothesis, we inserted microdialysis probes into the XII nucleus region of decerebrate, paralyzed, vagotomized and artificially ventilated cats. The probes were perfused without or with the addition of a 5HT reuptake blocker, clomipramine. The levels of 5HT and its metabolite, 5-hydroxyindoleacetic acid (5HIAA), were determined using HPLC and electrochemical detection in dialysate samples collected over successive 20 min periods under four successive experimental conditions: control (at least 2 h after probe insertion); during the postural atonia and respiratory depression produced by pontine microinjection of carbachol; recovery from the effects of carbachol produced by pontine microinjection of atropine; and, to verify that the presence of 5HT in the dialysate was related to the activity of serotonergic cells of the brainstem, following administration of 8-OH-DPAT, a 5HT 1A receptor agonist known to suppress activity in the serotonergic cells of the raphe system. After correcting for recovery rates of individual probes, the mean control 5HT level in the extracellular space of the XII nucleus region was 7.9 +/- 4.4 nM (S.D.) in eight experiments without reuptake blockers. During the carbachol-induced depression, it was reduced to 70 +/- 20% of the pre-carbachol level. It increased to the original control level 98 +/- 27% after pontine injection of atropine. 8-OH-DPAT reduced the 5HT level to 43 +/- 14% of the post-atropine level. Changes in the 5HIAA level were not as consistent as for 5HT and did not reach statistical significance under any of the experimental conditions. Thus, a functionally significant amount of 5HT is present in the extracellular space within the XII nucleus region, and its decrement during carbachol-induced, REM sleep-like atonia is likely to reflect that occurring during natural REM sleep; this may contribute to the decreased tone of upper airway muscles and airway patency.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1990

Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells.

The Ca2(+)-mobilizing actions of the muscarinic receptor agonist, methacholine (MeCh), and the microsomal Ca2+ pump inhibitor, thapsigargin, were investigated in lacrimal acinar cells. As previously shown for parotid cells (J. Biol. Chem. 264: 12266-12271, 1989), thapsigargin activates both internal Ca2+ release and Ca2+ entry from the extracellular space without increasing cellular inositol phosphates. The inorganic Ca2+ antagonist La3+ inhibited MeCh- or thapsigargin-activated Ca2+ entry. However, when added before MeCh or thapsigargin, La3+ inhibited the extrusion of Ca2+ at the plasma membrane. This phenomenon was exploited in protocols designed to investigate the pathways for filling agonist-sensitive Ca2+ stores in lacrimal cells. The results show that, in contrast to previous suggestions that external Ca2+ is required to replenish agonist-regulated Ca2+ stores, the inhibition of Ca2+ extrusion permits recycling of Ca2+ released by MeCh back into an MeCh- and thapsigargin-sensitive pool. Thus, although extracellular Ca2+ is the major source for refilling the intracellular Ca2+ stores under physiological conditions, the pathway by which this Ca2+ enters the pool need not be a direct one. These results are consistent with the recently revised capacitative model for the refilling of intracellular Ca2+ stores through Ca2+ influx subsequent to Ca2+ depletion, according to which refilling of intracellular Ca2+ stores occurs via a cytoplasmic route rather than a direct channel between intracellular Ca2+ stores and the extracellular space.

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