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[Vasorelaxant effects of forsythiaside from the fruits of Forsythia suspensa].

The vasorelaxant effects of forsythiaside (compound 1) from the fruits of Forsythia suspensa on isolated rat aortic rings were studied. Compound 1 showed a slow relaxation activity against norepinephrine (NE)-induced contractions of rat aorta with/without endothelium. This compound did not affect contractions induced by a high concentration of potassium (K(+) 60 mM), while it inhibited NE-induced vasocontraction in the presence of nicardipine. These results show that the inhibition by compound 1 of NE-induced vasocontraction is due to a decrease in calcium influx from the extracellular space caused by NE.

Animals↗

Spatio-temporal pattern of proliferation of immunoglobulin G-containing astrocytes in the injured mouse cerebral hemisphere.

Following a mechanical injury of the cerebral hemisphere in adult mice, [3H]thymidine was injected at different time intervals to reveal proliferating cells. Brain sections were double immunostained for immunoglobulin G (IgG) and glial fibrillary acidic protein (GFAP), and subjected to autoradiography. The procedure allowed to distinguish four cell types: (1) proliferating process-bearing cells immunopositive for IgG but expressing no GFAP (GFAP-/IgG+ PB cells) (2) proliferating and (3) non-proliferating astrocytes immunopositive both for GFAP and IgG (GFAP+/IgG+), (4) proliferating astrocytes expressing GFAP and containing no IgG (GFAP+/IgG-). Thereafter, the distribution of each cell type within the injury area was recorded at each stage of the experiment and displayed as contour line maps. During the whole posttraumatic period, areas of very strong proliferation of GFAP-/IgG+ PB-cells were located within deep layers of the cerebral cortex and in the white matter around the bottom part of the lesion. Few proliferating GFAP+/IgG+ astrocytes were mainly located in the same regions. However, changes in the distribution of proliferating astrocytes containing no IgG followed different spatio-temporal pattern. On the 4th day after injury areas of their proliferative activity surrounded both the bottom and superficial parts of the lesion. It seems that the immediate contact with the extravasated blood serum does not determine IgG cellular uptake. The uptake appears to depend on the cell type and its role in re-establishing the extracellular space in the injured brain tissue.

Animals↗

Further studies on the nature of fluid compartmentalization in chick calvaria.

Labeled mannitol and polyethylene glycol (mol. wt. 4000), PEG, were used as space markers to estimate the fluid compartmentalization of calvaria taken from 3-5-day-old chicks. The mannitol results indicated that about 80% of the water in these bones is extracellular. Only a small fraction of this extracellular space was available to the diffusion of polyehtylene glycol. Adsorption studies in vitro and prior incubation of the tissues with hydrolytic enzymes indicated that the phenomenon of exclusion of PEG was primarily physical in nature. The polyethylene polymer is excluded from the water of hydration of the mineral phase. Apparently much of the extracellular water (two-thirds) of bone is in the form of crystal hydration.

Adsorption↗

Thimerosal decreases TRPV1 activity by oxidation of extracellular sulfhydryl residues.

TRPV1, a receptor for capsaicin, plays a key role in mediating thermal and inflammatory pain. Because the modulation of ion channels by the cellular redox state is a significant determinant of channel function, we investigated the effects of sulfhydryl modification on the activity of TRPV1. Thimerosal, which oxidizes sulfhydryls, blocked the capsaicin-activated inward current (I(cap)) in cultured sensory neurons, in a reversible and dose-dependent manner, which was prevented by the co-application of the reducing agent, dithiothreitol. Among the three cysteine residues of TRPV1 that are exposed to the extracellular space, the oxidation-induced effect of thimerosal on I(cap) was blocked only by a point mutation at Cys621. These results suggest that the modification of an extracellular thiol group can alter the activity of TRPV1. Consequently, we propose that such a modulation of the redox state might regulate the physiological activity of TRPV1.

Animals↗

Imidazol-1-ylalkanoic acids as extrinsic 1H NMR probes for the determination of intracellular pH, extracellular pH and cell volume.

The synthesis and biological evaluation of a novel series of extrinsic probes for intracellular pH (pHi), extracellular pH (pHo) and cell volume determination by 1H NMR is described. Imidazol-1-ylacetate, malonate, 3-glutarate and 2-succinate esters were synthesized by reaction of imidazole with alpha-bromo esters or with alpha, beta-unsaturated esters. The corresponding acids were prepared by hydrolysis. Rat erythrocytes were readily permeable to methyl imidazol-1-ylacetate, moderately permeable to diethyl 2-imidazol-1-ylsuccinate and impermeable to diethyl 3-imidazol-1-yl-glutarate esters. Imidazol-1-ylacetic acid was the only acid derivative which penetrated the erythrocyte interior when added directly to the incubation medium. Transport of the permeable compounds to the erythrocyte interior was non-saturable up to 200 mM added compound. Addition of methyl imidazol-1-ylacetate or diethyl 2-imidazol-1-ylsuccinate esters to erythrocyte suspensions, resulted in hydrolysis to imidazol-1-ylacetic acid and 2-imidazol-1-ylsuccinic acid mono-ethyl ester in the intracellular and extracellular spaces, respectively. pHi and pHo were determined from the different chemical shifts of the H-2 proton of the acid derivatives in the intracellular (H-2i) and extracellular (H-2o) compartments. In addition, the relative intracellular and extracellular volumes could be calculated from the areas of the intracellular and extracellular H-2 resonances.

Animals↗

Failure of extracellular digestive enzymes to alter in vitro secretion by rat pancreas.

The basal rate of amylase secretion from rat pancreatic lobules was found to diminish with time. This decline, which is not due to depletion of amylase stores or loss of tissue integrity, has been attributed to the build-up of amylase in the extracellular space [Ho and Rothman, Am. J. Physiol. 245 (Cell Physiol. 14): C21-C27, 1983]. This accumulation of amylase would result in a decrease in the intracellular-extracellular amylase gradient, which, according to the so-called equilibrium hypothesis, is responsible for net amylase secretion under basal conditions. Using rat pancreatic lobules and acini, we have tested the validity of this hypothesis by adding to the incubation medium at the onset of incubation either collected secretory products or purified amylase. Based on the equilibrium hypothesis, one would have predicted that these additions would also reduce the concentration gradient favoring net secretion and would result in an apparent reduction in the initial rate of basal in vitro digestive enzyme secretion. The results obtained from these studies, however, were not in accord with those predictions, since these additions did not diminish the initial rate of basal amylase secretion. Our observations therefore indicate that the decrease in the rate of basal amylase secretion from rat pancreas with time is not due to the loss of a concentration gradient favoring efflux. These results argue against the validity of the equilibrium hypothesis.

Amylases↗

Studies on the kinetics of (3H)-ouabain uptake and exchange in the isolated papillary muscle of the guinea-pig.

1 The uptake, wash-out and exchange of [3H]-ouabain was studied in isolated, resting, and electrically stimulated papillary muscles of the guinea-pig. 2 At the equilibrium level of uptake, a different tissue/medium ratio was obtained for each of the concentrations used, i.e. 3.4, 1.8 and 0.82 for 1 times 10-minus 7M, 7.7 times 10-minus 7M, and 5 times 10-minus 6M ouabain, respectively. Equilibrium was reached more rapidly at high concentrations of ouabain. 3 The maximum number of binding sites for ouabain was estimated to be 1 times 10-15 binding sites/g wet weight. 4 No difference in [3H]-ouabain uptake could be detected between resting and electrically stimulated papillary muscles. 5 The kinetics of the ouabain uptake, wash-out and exchange are discussed. The results suggest that there is a saturable compartment in papillary muscle which can best be demonstrated if low concentrations of ouabain are used. Because of its small size, the saturable compartment submerges in the process of the filling of the extracellular space at high concentrations (5 times 10-minus 6M).

Animals↗

The effect of calcium on the lens ultrastructure.

The influence of a calcium-enriched solution on pig lens cell structure was studied in vitro. The morphological changes which occurred 20 min or 2 h after bathing in the solution were studied by transmission electron microscopy. After 20-min incubation, small intercellular vacuoles containing membranous and amorphous substance occurred mainly in the epithelial cells. The cytoplasm of the cortical fibers had already been dissolved at this stage. Here the membrane was disrupted and the extracellular spaces widened. After 2-h incubation, the epithelial cells were compressed by adjacent swollen fibers. However, vacuolization in the epithelium had decreased at this stage. At the cortical fiber zone, vacuolar formation had increased extensively. Vacuolization was mainly observed in the intercellular spaces between the intact gap junctions. After 2 h, the main alterations were membrane disruptions and advancing dissolution of the cytoplasm. Deeper cortical fibers were swollen but showed no extracellular vacuoles.

Actin Cytoskeleton↗

Extracellular TNFR1 release requires the calcium-dependent formation of a nucleobindin 2-ARTS-1 complex.

Extracellular tumor necrosis factor (TNF) receptors function as TNF-binding proteins that modulate TNF activity. In human vascular endothelial cells (HUVEC), extracellular TNFR1 (type I TNF receptor, TNFRSF1A) is generated by two mechanisms, proteolytic cleavage of soluble TNFR1 ectodomains and the release of full-length 55-kDa TNFR1 in the membranes of exosome-like vesicles. TNFR1 release from HUVEC is known to involve the association between ARTS-1 (aminopeptidase regulator of TNFR1 shedding), an integral membrane aminopeptidase, and TNFR1. The goal of this study was to identify ARTS-1 binding partners that modulate TNFR1 release to the extracellular space. A yeast two-hybrid screen of a human placenta cDNA library showed that NUCB2 (nucleobindin 2), via its helix-loop-helix domains, binds the ARTS-1 extracellular domain. The association between endogenous ARTS-1 and NUCB2 in HUVEC was demonstrated by co-immunoprecipitation experiments, which showed the formation of a calcium-dependent NUCB2.ARTS-1 complex that associated with a subset of total cellular TNFR1. Confocal microscopy experiments demonstrated that this association involved a distinct population of NUCB2-containing intracytoplasmic vesicles. RNA interference was utilized to specifically knock down NUCB2 and ARTS-1 expression, which demonstrated that both are required for the constitutive release of a full-length 55-kDa TNFR1 within exosome-like vesicles as well as the inducible proteolytic cleavage of soluble TNFR1 ectodomains. We propose that calcium-dependent NUCB2.ARTS-1 complexes, which associate with TNFR1 prior to its commitment to pathways that result in either the constitutive release of TNFR1 exosome-like vesicles or the inducible proteolytic cleavage of TNFR1 ectodomains, play an important role in mediating TNFR1 release to the extracellular compartment.

Aminopeptidases↗

Kinetics of amino acid transport across bone marrow cell membranes.

Dog bone marrow nucleated cells were incubated in media containing labeled L-amino acids, and the cellular accumulation of radioactivity as a function of time was measured and analyzed according to a three-compartment model.(a) The turnover half-time of intracellular histidine arising from extracellular sources was 6.0 +/-0.7 (SEM) min. Similar turnover half-time for serine was 10 +/-2 (SEM) min; for tryptophan, 6.5 +/-1.2 (SEM) min; and for methionine, 4.4 +/-0.6 (SEM) min. Loss of the intracellular amino acids to the extracellular space accounted for the major portion of their turnover.(b) Each of the four amino acids noted above appeared to be actively transported into the cell.(c) At physiologic extracellular histidine concentrations, histidine entered the cell predominantly by a facilitated process with an apparent Michaelis constant of 0.28 mmole/liter and a limiting flux of 14 x 10(-8) mmumole/min per cell. Loss of histidine from the cell appeared to be substantially facilitated with an apparent Michaelis constant greater than that for histidine entry.(d) Insulin and glucagon had no measurable effect on histidine transport across the bone marrow cell membrane.(e) Methionine depressed the influx and the fractional turnover rate of the intracellular pool of both histidine and serine.(f) The extent of cellular accumulation of alpha-N-formiminoglutamate and alpha-N-formylglutamate was about 1/100 that of histidine. alpha-N-formiminoglutamate added to the culture was about (1/4) as effective as histidine in providing monocarbon fragments for DNA thymine synthesis.

Amino Acids↗

Ultrastructural localization of endogenous calcium in the teleost retina.

The ultrastructural localization of endogenous calcium in the retina of adult cichlid fish Oreochromis mossambicus (Teleostei) was studied using the cytochemical osmiate-bichromate method of Probst (1986). The specificity of this method for calcium localization was proven by means of EGTA treatment of ultrathin sections and electron-spectroscopic-imaging technique (ESI) with an energy-filtering transmission electron microscope (CEM 902, Zeiss). Large amounts of electron-dense calcium containing deposits were found in the outer segments of rods, in the synaptic vesicles of receptor terminals and bipolar cells, in the perinuclear space of photoreceptors and in the endoplasmic reticulum of different cell types, especially in the inner segment and fibres of photoreceptor cells. In the inner plexiform layer calcium was detected in the extracellular space with greater accumulations in the synaptic cleft. Principal differences in the localization of calcium between rods and cones and between several types of synapses and vesicles are shown. The possible role of calcium in the subcellular structures of retinal cells is discussed.

Animals↗

Ultrastructure of the interface between cultured osteoblasts and surface-modified polymer substrates.

Osteoblasts derived from rat bone marrow cells were cultured on surface-modified poly(ethylene terephthalate) films in the presence of ascorbic acid, beta-glycerophosphate, and dexamethasone. The surfaces employed for cell culture included the untreated hydrophobic surface and three modified surfaces possessing immobilized phosphate polymer chains, collagen molecules, and a thin hydroxyapatite-deposited layer. They all were produced by photo-induced graft polymerization with subsequent surface modifications of the graft chains. The ultrastructural morphology of the substrate/cell interfaces formed in in vitro osteoblast culture on these substrates was studied by transmission electron microscopy. The osteoblasts cultured for 1 week on the modified surfaces showed rough endoplasmic reticula rich in intracellular space and early matrix production in the extracellular space, irrespective of the surface chemistry. After 2 weeks of culture, osteoblasts exhibited active elaboration of extracellular matrix proteins, mostly composed of collagen, on all the surfaces. A remarkable result observed at this stage was direct deposition of an electron-dense, afibrillar layer of 180 nm thickness onto the surface having phosphate polymer chains. This layer became much more electron dense after 3 weeks of culture. Energy dispersive X-ray microanalysis revealed the presence of calcium phosphate in this layer. It was further found that the predeposited hydroxyapatite layer on the phosphate polymer-grafted surface promoted mineral deposition in the extracellular matrix that surrounded cuboid, osteocyte-like cells.

Animals↗

Blood-brain interfaces in vertebrates: a comparative approach.

The neuronal microenvironment in the vertebrate brain is isolated from plasma by a series of selective membranes, including the blood-brain barrier, the choroid plexus, and the meningeal barrier. This review deals with the structure and function of these selective membranes in the different vertebrate classes. Present knowledge indicates that all vertebrates have brain barrier membranes and, further, that functional characteristics of these membranes are basically similar in all the vertebrate classes. The blood-brain barrier (or capillary-glial complex) and the meningeal barrier have many of the properties of a tight epithelium, including the presence of tight junctions and specific transport mechanisms. The choroidal epithelium is a typical secretory epithelium. The functional significance of the specialized membranes located at the blood-brain interface is considered, and we suggest that the phylogenetic development of a blood-brain barrier provided neurons of the vertebrate brain with a unique extracellular milieu optimal both for synaptic communication and for nonsynaptic communication via the entire extracellular space.

Adaptation, Physiological↗

[Imbalances of the water and electrolyte status].

Nutritional therapy can be impaired if imbalances in water and electrolyte status have led to gross disorders of the cardiovascular, pulmonary, renal, metabolic, and central nervous systems. Restauration and maintenance of the functional extracellular fluid volume is the primary therapeutic goal in water and electrolyte resuscitation. Hyper- and hypoosmolar disturbances are automatically corrected by intrinsic regulatory mechanisms. Potassium deficiency or overload, or potassium disequilibrium between the intracellular and extracellular space can lead to dangerous cardiac arrhythmias. Hyper- and hypokalemia usually develop within days or even weeks and should not be corrected within a few hours. If life threatening hyperkalemia develops during acute renal failure, 20 ml 10% calcium gluconate solution can be given intravenously in order to avoid ventricular fibrillation or cardiac arrest. The discrimination between prerenal disease, acute tubular necrosis and other causes of acute renal failure is based on special investigations, such as urinary osmolality, urinary sodium concentration, clearance of creatinine, osmolar solutes, free water, and fractional sodium excretion. The clinical examination of a patient should be the basis of assessing his water and electrolyte state. Laboratory findings which are in disagreement with the clinical state have to be repeated, critically interpreted, but not completely rejected. Third space losses make fluid balance difficult.

Acid-Base Equilibrium↗

Dexamethasone treatment during hemorrhagic shock: changes in extracellular fluid volume and cell membrane transport.

Changes in extracellular fluid volume and cell membrane transport during hemorrhagic shock and the effects of dexamethasone treatment on these changes were measured. It is well known that prolonged hemorrhagic shock leads to irreversible changes and a progressive decrease in blood pressure despite reinfusion for lost blood. Pharmacologic doses of glucocorticoids provide some protection against these changes. Therefore, one purpose in the present study was to identify possible sites of glucocorticoid action whicy may prevent the irreversible changes from occurring. The extracellular fluid volume in normal control, nontreated dogs in shock, and dexamethasone-treated dogs in shock were measured by a dilution technique, using [35S] sodium sulfate. Cell membrane cation transport capabilities were measured in liver slices, diaphragm slices, and red blood cells taken from normal control, nontreated rats in shock, and dexamethasone-treated rats in shock. The accumulation of radioactivity by the tissues incubated with 22Na served as an indicator of cell membrane ion transport capabilities. The results indicate that in animals subjected to prolonged hemorrhagic shock, there is a fluid shift from the extracellular space into intracellular spaces, reducing blood volume. Cell membranes are damaged and transport mechanisms are altered; therefore, the cells are unable to extrude ions along with water. Dexamethasone treatment was shown to prevent extracellular fluid volumes from decreasing below that amount due to the plasma lost during hemorrhage. Also, it prevented some cell membrane damage and maintained membrane transport mechanisms near normal. In addition, at the onset of dexamethasone injection, blood pressure increased, and urine output was restored.

Animals↗

Assessment of extracellular glucose distribution and glucose transport activity in conscious rats.

The effects of insulin on extracellular glucose distribution and cellular glucose transport activity were studied by simultaneously analyzing the plasma kinetics of L-[1-14C]glucose and 3-O-[3H]methylglucose after an intravenous injection during saline or insulin infusion (euglycemic glucose clamp) in conscious rats (n = 7 for each). The time profiles of plasma L-glucose were almost superimposable in the two protocols, and compartmental analysis showed that neither distribution volumes nor distribution rate constants were affected with insulin (P > 0.05 for all), suggesting that glucose distribution within the extracellular space was not influenced with insulin. In contrast, the time profile of plasma 3-O-methylglucose (3-MG) was markedly altered with insulin; the initial decrease was much faster during insulin infusion than during saline infusion, indicating stimulation of 3-MG transport into intracellular spaces with insulin. The 3-MG data were analyzed using a comprehensive model separately describing extracellular distribution and cellular transport of 3-MG by incorporating information on extracellular distribution kinetics obtained from L-glucose data. The combined L-glucose and 3-MG kinetic analysis precisely estimated insulin's effect in vivo to stimulate glucose transport into and out of intracellular spaces. We conclude that 1) insulin does not affect extracellular glucose distribution kinetics or volumes in conscious rats and 2) insulin's effects on cellular glucose transport in vivo can be assessed by simultaneous analysis of plasma L-glucose and 3-MG kinetics.

3-O-Methylglucose↗

Interpretation of DW-NMR data: dependence on experimental conditions.

This review examines the effect of experimental conditions on the data obtainable from diffusion weighted NMR experiments. The origin and forms of the Stejskal-Tanner experiment are presented, and the relative merits of bipolar to monopolar diffusion weighting gradient pulses are discussed, as are those of spin-echo and stimulated-echo weighting schemes. The short pulse Stejskal-Tanner experiment as required for q-space imaging is described. Criteria for successful diffusion weighted imaging are given, and current strategies for diffusion weighted imaging are evaluated against these. The range of biological objects accessible to diffusion weighted NMR is summarized, together with the associated experimental limitations. In the final section the dependence of diffusion weighted NMR data on diffusion time and b-value range is examined, and the relationship between apparent restricted diffusion and the size of the extracellular space is demonstrated.

Diffusion↗

Use of microdialysis to study interstitial nitric oxide and other reactive oxygen and nitrogen species in skeletal muscle.

Microdialysis techniques can be used to sample the interstitial space of tissues such as skeletal muscle. Analytical developments have allowed adaptations of these techniques to permit continuous monitoring of nitric oxide and a number of other reactive oxygen and nitrogen species in skeletal muscle extracellular space. Methods are described for assessment of interstitial nitrate and nitrite content, superoxide anion content, hydroxyl radical activity, and the content of relatively stable lipid radicals detectable using spin trapping and electron spin resonance techniques in skeletal muscle of rodents at rest and during contractile activity.

Animals↗