Blockade of the Noradrenaline Carrier Increases Extracellular Dopamine Concentrations in the Prefrontal Cortex: Evidence that Dopamine Is Taken up In Vivo by Noradrenergic Terminals

The effect of systemic administration of desmethylimipramine (DMI) and oxaproptiline (OXA), two inhibitors of the noradrenaline (NA) reuptake carrier, on the in vivo extracellular concentrations of dopamine (DA) was studied by transcerebral dialysis in the prefrontal cortex and in the dorsal caudate of freely moving rats. In the NA-rich prefrontal cortex, either drug increased extracellular DA concentrations whereas in the dorsal caudate neither was effective. Haloperidol increased extracellular DA concentrations more effectively in the dorsal caudate than in the prefrontal cortex. Pre-treatment with DMI or OXA, which failed to modify the effect of haloperidol in the dorsal caudate, potentiated its action in the prefrontal cortex. 6-Hydroxydopamine lesioning of the dorsal NA bundle prevented the ability of OXA to increase DA concentrations. The results suggest that reuptake into NA terminals in an important mechanism by which DA is cleared from the extracellular space in a NA-rich area such as the prefrontal cortex. The elevated extracellular concentrations of DA resulting from blockade of such mechanism by tricyclic antidepressants may play a role in the therapeutic effects of these drugs.     

The volume of extracellular fluid under conditions of long-term space flights

The volume of extracellular fluid (the bromine space) was determined in 18 cosmonauts 30 days before the start of a space flight and on the first day after landing. The duration of space flights on the Mir orbital station was from 126 to 438 days. Moreover, the volume of extracellular fluid was determined in seven cosmonauts directly during long-term space flights approximately two weeks before landing. After long-term space flights, the volume of extracellular fluid was decreased in all cosmonauts studied. The bromine space volume was significantly decreased compared to its initial preflight value. A decrease in the volume of extracellular fluid was caused not only by the reduction in the dense mass of the body but also by its dehydration. These processes developed independently of the duration of weightlessness but are mainly determined by the individual features of human beings.     

Extracellular trafficking of myocilin in human trabecular meshwork cells

Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.     

Catalytic transitions in the human MDR1 P-glycoprotein drug binding sites

Multidrug resistance proteins that belong to the ATP-binding cassette family like the human P-glycoprotein (ABCB1 or Pgp) are responsible for many failed cancer and antiviral chemotherapies because these membrane transporters remove the chemotherapeutics from the targeted cells. Understanding the details of the catalytic mechanism of Pgp is therefore critical to the development of inhibitors that might overcome these resistances. In this work, targeted molecular dynamics techniques were used to elucidate catalytically relevant structures of Pgp. Crystal structures of homologues in four different conformations were used as intermediate targets in the dynamics simulations. Transitions from conformations that were wide open to the cytoplasm to transition state conformations that were wide open to the extracellular space were studied. Twenty-six nonredundant transitional protein structures were identified from these targeted molecular dynamics simulations using evolutionary structure analyses. Coupled movement of nucleotide binding domains (NBDs) and transmembrane domains (TMDs) that form the drug binding cavities were observed. Pronounced twisting of the NBDs as they approached each other as well as the quantification of a dramatic opening of the TMDs to the extracellular space as the ATP hydrolysis transition state was reached were observed. Docking interactions of 21 known transport ligands or inhibitors were analyzed with each of the 26 transitional structures. Many of the docking results obtained here were validated by previously published biochemical determinations. As the ATP hydrolysis transition state was approached, drug docking in the extracellular half of the transmembrane domains seemed to be destabilized as transport ligand exit gates opened to the extracellular space.     

Myocardial capillaries in the fetal and the neonatal rat: a morphometric analysis of the maturing myocardial capillary bed

Developing myocardial capillaries from 16-day-gestation fetus to adult undergo several morphological changes including a thinning of the lateral extensions of the capillary endothelial cells, the formation of a basal lamina, and an increase in the number of plasmalemmal vesicles. A decrease in the extracellular space, an increase in the number of capillaries, and a decrease in the capillary diameter were also observed during the developmental period. In view of these ultrastructural changes, a morphometric analysis was made on the developing myocardial wall to demonstrate specific quantitative changes. The volumes which were occupied by capillary endothelial cells, capillary lumina, extracellular space, and myocardial myocytes within a reference volume of myocardium were measured; and we found that 8% of the reference myocardial volume was occupied by capillary endothelial cells, 85% was occupied by myocardial myocytes, 4% was occupied by capillary lumina, and, except for a significant change in extracellular space at 16 days gestation, 3% was occupied by extracellular space. Each volume ratio was found to be nearly constant throughout the studied period. In contrast to this constancy in the volume ratios, other parameters which were measured demonstrated significant changes during the developmental period studied. These overall changes include a 135% increase in capillary density, a 63% increase in luminal surface area of capillary endothelial cells, a 24% decrease in capillary diameter, a 12% decrease in diffusion distance, and a 35% decrease in the diameter of the erythrocyte population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusion from gel in brain: Modelisation and identification

A mathematical model is proposed for describing the mechanism of diffusion from gel (Tissucol) into the extracellular space. After diffusion of the antibiotic in one dimension, the gradient concentration was determined with microvoltametric electrodes. These microelectrodes measure the free diffusible form of electroactive antibiotics in the extracellular brain space. The aim of this study was to find simultaneously the coefficient of diffusion and extraction of some antibiotics (in our case the Fotemustin) using the Alienor Algorithm. These coefficients are useful for predicting the concentration gradient into abscesses, fibrin, etc.     

D-[1-14C]mannitol and [U-14C]sucrose as extracellular space markers for human spermatozoa and the uptake of 2-deoxyglucose

[U-14C]Sucrose and D-[1-14C]mannitol were used to determine the tritiated water space of human spermatozoa to validate these compounds as markers for the extracellular space. Calculations based on 0.03 mM-[U-14C]sucrose gave a negative water space. The water space estimated with 0.03 mM-D[1-14C]mannitol was unstable but a stable result was obtained with 0.3 mM-D-[1-14C]mannitol in incubations up to 2 h. The mean water space was 2.21 +/- 0.106 microliters/10(8) spermatozoa (mean +/- s.e.m. for 6 batches of pooled semen). The water space was decreased or abolished by Triton X-100, cold shock, sonication or hypotonic treatment. The water space responded to changes in the osmolarity of the medium by increasing in dilute media. It is concluded that mannitol is an effective extracellular marker for human spermatozoa if concentrations greater than or equal to 0.3 mM are used. When the kinetics of the uptake of 2-deoxyglucose by the spermatozoa were studied by using mannitol as an extracellular marker, the transport was saturable and was inhibited by cytochalasin B. The Km was 1.6 +/- 0.33 mM and the Vmax was 4.2 +/- 0.52 nmol/10(8) spermatozoa/10 sec (mean +/- s.e.m., n = 4).     

pH regulation in the stomatogastric ganglion of the crab Cancer pagurus

The regulation of intracellular neuronal pH and pH from the extracellular space was studied in the isolated stomatogastric ganglion of the crab Cancer pagurus. Intracellular neuronal pH was found to be 0.3–0.4 pH units more acidic than the standard bath pH of 7.4 and surprisingly, the extracellular space pH was found to be around 0.1 pH units more alkaline than the bath pH. Extracellular space pH shifts in response to bath pH changes decreased as a function of the depth of the recording site within the ganglion, suggesting the existence of restrictions in the free diffusion of H⁺. The amplitude of these pH_e shifts increased in Na⁺-free saline or with amiloride, suggesting Na⁺-dependent regulation of the extracellular space pH. In Na⁺ free saline or in the presence of amiloride, intracellular pH recovery from an NH₄Cl induced acidosis was reduced, and the H⁺ muffling capacity (cf. Thomas et al. 1991) of the extracellular space was markedly reduced. Changes of bath pH had only small effects on the rhythm generating properties of one of the central pattern generators of the stomatogastric ganglion, while NH₄Cl-induced intraganglionic pH changes markedly altered this rhythm.Abbreviations CPG central pattern generator - ECS extracellular space - LP lateral pyloric neuron - NMDG N-methyl-D-glucamine - PD pyloric dilator neuron - pH_e extracellular space pH - pH_i intracellular pH - pH_o bath pH - STG stomatogastric ganglion - V_ref reference potential     

Calcium and pancreatic beta-cell function: glucose stimulation uptake of lanthanum-displaceable 45-Ca from low or normal calcium-containing media.

The uptake of 45Ca was studied in beta-cell-rich pancreatic islets microdissected from ob/ob-mice. Glucose stimulated 45Ca incorporation in a lanthanum-displaceable pool remaining after correction for the extracellular space occupied by sucrose. It is suggested that the glucose effect on the lanthanum-displaceable calcium is the cause rather than the result of the secretion of insulin since it could be demonstrated also in the presence of only trace amounts of extracellular calcium.     

Potassium modulation of taurine transport across the frog retinal pigment epithelium

Net taurine transport across the frog retinal pigment epithelium-choroid was measured as a function of extracellular potassium concentration, [K+]o. The net rate of retina-to-choroid transport increased monotonically as [K+]o increased from 0.2 mM to 2 mM on the apical (neural retinal) side of the tissue. No further increase was observed when [k+]o was elevated to 5 mM. The [K+]o changes that modulate taurine transport approximate the light-induced [K+]o changes that occur in the extracellular space separating the photoreceptors and the apical membrane of the pigment epithelium. The taurine-potassium interaction was studied by using rubidium as a substitute for potassium and measuring active rubidium transport as a function of extracellular taurine concentration. An increase in apical taurine concentration, from 0.2 mM to 2 mM, produced a threefold increase in active rubidium transport, retina to choroid. Net taurine transport can also be altered by relatively large, 55 mM, changes in [Na+]o. Apical ouabain, 10(-4) M, inhibited active taurine, rubidium, and potassium transport; in the case of taurine, this inhibition is most likely due to a decrease in the sodium electrochemical gradient. In sum, these results suggest that the apical membrane contains a taurine, sodium co-transport mechanism whose rate is modulated, indirectly, through the sodium pump. This pump has previously been shown to be electrogenic and located on the apical membrane, and its rate is modulated, indirectly, by the taurine co-transport mechanism.     

Identification of phosphorylation sites on extracellular corneal epithelial cell maspin

Maspin, a 42-kDa non-classical serine protease inhibitor (serpin), is expressed by epithelial cells of various tissues including the cornea. The protein localizes to the nucleus and cytosol, and is present in the extracellular space. While extracellular maspin regulates corneal stromal fibroblast adhesion and inhibits angiogenesis during wound healing in the cornea, the molecular mechanism of its extracellular functions is unclear. We hypothesized that identifying post-translational modifications of maspin, such as phosphorylation, may help decipher its mode of action. The focus of this study was on the identification of phosphorylation sites on extracellular maspin, since the extracellular form of the molecule is implicated in several functions. Multi-stage fragmentation MS was used to identify sites of phosphorylation on extracellular corneal epithelial cell maspin. A total of eight serine and threonine phosphorylation sites (Thr50, Ser97, Thr118, Thr157, Ser240, Ser298, Thr310 and Ser316) were identified on the extracellular forms of the molecule. Phosphorylation of tyrosine residues was not detected on extracellular maspin from corneal epithelial cell, in contrast to breast epithelial cells. This study provides the basis for further investigation into the functional role of phosphorylation of corneal epithelial maspin.     

外源钙调素与拟南芥悬浮培养细胞结合位点的观测及其胞外效应

以拟南芥悬浮培养细胞为实验体系,借助外源荧光及同位素标记钙调素,研究结果表明外源钙调素不能被主动内吞入细胞内,而是主要以完整分子形式结合在细胞外表面;外源纯化钙调素可促进正向型质膜囊泡中的鸟苷酸三磷酸水解酶活性升高,也可引起拟南芥悬浮细胞质游离钙离子浓度的特异升高,表明外源钙调素可能通过细胞表面位点跨膜信号转换为细胞内信号,从而调节生物学活性。     

The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells without a Signal Peptide at Hand

For a long time, protein transport into the extracellular space was believed to strictly depend on signal peptide-mediated translocation into the lumen of the endoplasmic reticulum. More recently, this view has been challenged, and the molecular mechanisms of unconventional secretory processes are beginning to emerge. Here, we focus on unconventional secretion of fibroblast growth factor 2 (FGF2), a secretory mechanism that is based upon direct protein translocation across plasma membranes. Through a combination of genome-wide RNAi screening approaches and biochemical reconstitution experiments, the basic machinery of FGF2 secretion was identified and validated. This includes the integral membrane protein ATP1A1, the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), and Tec kinase, as well as membrane-proximal heparan sulfate proteoglycans on cell surfaces. Hallmarks of unconventional secretion of FGF2 are: (i) sequential molecular interactions with the inner leaflet along with Tec kinase-dependent tyrosine phosphorylation of FGF2, (ii) PI(4,5)P₂-dependent oligomerization and membrane pore formation, and (iii) extracellular trapping of FGF2 mediated by heparan sulfate proteoglycans on cell surfaces. Here, we discuss new developments regarding this process including the mechanism of FGF2 oligomerization during membrane pore formation, the functional role of ATP1A1 in FGF2 secretion, and the possibility that other proteins secreted by unconventional means make use of a similar mechanism to reach the extracellular space. Furthermore, given the prominent role of extracellular FGF2 in tumor-induced angiogenesis, we will discuss possibilities to develop highly specific inhibitors of FGF2 secretion, a novel approach that may yield lead compounds with a high potential to develop into anti-cancer drugs.     

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport

The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.     

The Dimensions of the Extracellular Space in Sartorius Muscle

A survey has been made of the amount of muscle water available to inulin, sucrose, and radioiodinated human serum albumin (RISA). The percentage spaces available to the three molecules are of the same order of magnitude, but the sucrose space > inulin space > albumin space. The kinetics of influx and efflux of RISA have been studied, and it appears that a small part of the albumin may be adsorbed in the extracellular phase. Nevertheless the albumin space would appear to give the best index of the extracellular volume. The scatter in values found for the extracellular space by all methods is very great, ranging from 8 to 40 per cent and renders invalid the use of a mean value for the calculation of intracellular concentrations. The variation within paired muscles is less than between pairs, provided the tissue has undergone no volume change. Increase in total muscle volume when the muscle is placed in a hypotonic solution leads to a decrease in the size of the extracellular space.     

Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.     

Brain interstitial fluid drainage and extracellular space affected by inhalational isoflurane: in comparison with intravenous sedative dexmedetomidine and pentobarbital sodium

Brain interstitial fluid drainage and extracellular space are closely related to waste clearance from the brain. Different anesthetics may cause different changes of brain interstitial fluid drainage and extracellular space but these still remain unknown. Herein,effects of the inhalational isoflurane, intravenous sedative dexmedetomidine and pentobarbital sodium on deep brain matters' interstitial fluid drainage and extracellular space and underlying mechanisms were investigated. When compared to intravenous anesthetic dexmedetomidine or pentobarbital sodium, inhalational isoflurane induced a restricted diffusion of extracellular space, a decreased extracellular space volume fraction, and an increased norepinephrine level in the caudate nucleus or thalamus with the slowdown of brain interstitial fluid drainage. A local administration of norepinephrine receptor antagonists, propranolol,atipamezole and prazosin into extracellular space increased diffusion of extracellular space and interstitial fluid drainage whilst norepinephrine decreased diffusion of extracellular space and interstitial fluid drainage. These findings suggested that restricted diffusion in brain extracellular space can cause slowdown of interstitial fluid drainage, which may contribute to the neurotoxicity following the waste accumulation in extracellular space under inhaled anesthesia per se.     

The interphotoreceptor space. I. Postnatal ontogeny in mice and rats

The postnatal ontogeny of the retinal interphotoreceptor space of mice and rats was studied by electron microscopy to elucidate the cytological developments in the surrounding cells relevant to the accumulation of extracellular glycosaminoglycans and glycoproteins constituting the interphotoreceptor matrix. This extracellular material at birth is principally the cell coat on all the immature cells that border the space at that time, but later additional weblike strands are seen in the space. The cells delimiting the space in the adult are the pigment epithelium (PE), the photoreceptors, and the glial cells of Müller. The Golgi complex of the PE is large at birth but involutes by day 15. Melanogenesis is the principal activity in this cell at birth but as the melanin granules mature, lysosomes gradually accumulate. Growth of the apical microvilli of the PE is most pronounced between day 5 and 15, which is also the time of rapid expansion of the interphotoreceptor space. The Golgi complex of the photoreceptor enlarges during this interval also, and remains voluminous thereafter. Müller's cells insert only slender apical processes lacking in secretory vesicles, into the interphotoreceptor space. All the adult cells have a cell coat on the surfaces facing the interphotoreceptor space. Secretory vesicles were not identified in any of the cells impinging on the space.     

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

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