L-threodops increases extracellular norepinephrine levels in the brain: an in vivo study.

Extracellular norepinephrine levels in the striatum of normal rats increased significantly following the administration of L-threodops. When peripheral decarboxylase activity was inhibited with carbidopa, administration of the same dose of L-threodops induced a 2.5-fold greater increase in norepinephrine levels.

Interstitial albumin-bound dye concentrations in mice with a protein-rich effusion.

Levels of intravenously injected Evans blue dye in eluates of the lung and kidney, an index of interstitial fluid albumin concentration, together with water content of these tissues and levels of serum albumin were measured in Ha-icr mice with a tumor cell-induced protein-rich peritoneal effusion. By the fourth day after the intraperitoneal injection of tumor cells, when mean serum albumin levels had fallen to 76% of control values, mean albumin bound dye concentrations in lung and kidney had decreased to 63 and 58%, respectively, of control values. By the tenth day when serum albumin levels had decreased further to 67% of control values, albumin-bound dye concentrations in the lung and kidney had decreased to 58 and 43%, respectively, of control values. During this 10-day period the water content of the lung remained unchanged whereas that of the kidney had decreased by 7%. These observations suggest that the reduction in serum albumin which results from an abnormal distribution of this protein into a nonvascular compartment is accompanied, as in other models of hypoalbuminemia, by a more than proportionate reduction in interstitial albumin concentration in the lung and kidney.

[Problems of muscular fatigue--relationship to stimulation conduction velocity and K(+) concentration]. / Probleme der Muskelermüdung--Beziehung zur Erregungsleitungsgeschwindigkeit und K(+)-Konzentration.

Muscle fatigue is accompanied by a series of biochemical correlations as substrate depletion, lactate accumulation, shifts of pH, increase of phosphate (Pi), arise of free radicals or disturbances of ionic balances. In last time high interest has been directed to the increase of extracellular potassium during extensive muscle activity. It was suggested that high K+ concentration in the interstitium may alter propagation of action potential along the T-tubules or induces membrane depolarization with physiological consequences. In order to elucidate the role of potassium accumulation, experiments were performed on isolated rat muscles. An elevation from 5 to 10 mmol K+ of the bath solution causes a significant decrease of the conduction velocity of the action potential. This effect is more pronounced on fatigue-sensitive fast twitch EDL muscles than on fatigue-resistant slow twitch SOL muscles. Moreover, after tetanic stimulations of these muscles in normal solution, the conduction velocity dropped by the same amount as in high K+ solution but, again, differently in both muscle types. Therefore it is supposed that K+ accumulation during intensive muscle activity contributes to fatigue.

Enhanced neuroinhibitory effect of diltiazem in blood vessels of spontaneously hypertensive rats.

This study investigated the effects of diltiazem (a Ca2(+)-entry blocker) on neuromuscular junctions of blood vessels in hypertension. In isolated perfused mesenteric vasculatures prepared from spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats (WKY), the effects of diltiazem on norepinephrine release from vascular adrenergic neurons and pressor responses were examined. The influences of extracellular Ca2(+)-reduction on these responses were also studied. Stimulation evoked pressor responses and norepinephrine release were significantly greater in the mesenteric vasculatures of SHR than in those of WKY. Diltiazem inhibited both pressor responses and norepinephrine release during electrical nerve stimulation in a dose-dependent manner. The suppression of these responses was more pronounced in SHR than in WKY. Reduction of extracellular Ca2(+)-concentration also decreased the responses in SHR and WKY, and the inhibitory degree was significantly greater in SHR than in WKY. These results demonstrate that diltiazem affected the presynaptic site of the mesenteric vasculatures and decreased the stimulation-evoked norepinephrine release from vascular adrenergic neurons with a concomitant reduction of pressor responses of the preparation. Furthermore, the marked inhibition of pressor responses and norepinephrine release by diltiazem or Ca2(+)-depletion in SHR may suggest the increased Ca2(+)-dependency in vascular neurotransmission in this model of hypertension.

[Structure of the interstitial space and ground substance of the human articular cartilage]. / Struktura interstitsial'nogo prostranstva i osnovnogo veshchestva sustavnogo khriashcha cheloveka.

By means of transmissive and scanning electron microscopy (investigation of ultra-replicas) three-dimensional organization of the interstitial (interfibrillar) space of the articular cartilage has been demonstrated; it repeats, to some extent, construction of the fibrous base. By means of mercury porometry quantitative characteristics of various parameters of the interfibrillar space are obtained. Their specific volume is 0.96 cm3/g of the dehydrated cartilage, space with equivalent diameters from 300 up to 5 nm makes 94%. By means of the gas adsorption method it has been stated that the specific internal surface is 23.8 m2 per 1 g of the dehydrated articular cartilage. Transmissive and scanning electron histochemistry has revealed several various forms of structured proteoglycans, demonstrated their spatial organization and interconnection with collagenous fibrils. The methodical complex applied can be used for investigating the connective tissue interstitial spaces in other parts of the human locomotor apparatus.

Detection of extracellular calcium gradients with a calcium-specific vibrating electrode.

We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase-sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.

Microdialysis--a novel technique for clinical investigations.

Microdialysis is a novel, non-traumatic technique which, when properly performed, allows continuous direct measurements of substances in the interstitial space of a tissue or organ. It also makes it possible to obtain protein-free fluid for analyses, while contaminating bacteria or viruses are excluded. The measurements provide us with an insight into metabolic and pharmacological events at the cellular level in a target organ. Apart from its usefulness for experimental work, it is likely to become an important tool for defined areas in clinical practice.

Cyclosporin A levels in suction-blister fluid of patients with psoriasis treated systemically.

Oral cyclosporin A (CyA) is highly effective in the treatment of psoriasis. The long-term use is limited by dose-dependent side-effects, and the local concentration of CyA is a determining factor in treatment. The concentration of CyA in suction-blister fluid (SBF) and in whole blood was assessed using a polyclonal radioimmunoassay (RIA). This was carried out in patients with psoriasis following a single dose of CyA and while on adequate oral treatment with the drug. Peak blister fluid levels ranged from 32 to 170 ng/ml, and whole blood levels from 1250 to 2540 ng/ml. CyA trough levels (12 h following taking the drug) in the blister fluid ranged from 22 to 113 ng/ml, and in whole blood from 148 to 935 ng/ml. The trough concentrations in SBF were approximately 10% of whole blood trough levels.

Effect of phosphate on antibiotic and extracellular protein production by Myxococcus coralloides D.

The effect of inorganic phosphate concentrations on antibiotic and extracellular protein production by Myxococcus coralloides D have been examined. Antibiotic production by growing cells of this myxobacterium was maximal at phosphate concentrations of 10-20 mM, but was inhibited by concentrations higher than 20 mM. The total extracellular protein and the extracellular protein per cell ratio were independent of phosphate levels in the culture broth.

Measurement of kynurenic acid in mammalian brain extracts and cerebrospinal fluid by high-performance liquid chromatography with fluorometric and coulometric electrode array detection.

Kynurenic acid is a broad-spectrum excitatory amino acid (EAA) receptor antagonist which is present in the mammalian central nervous system. We describe a method for the measurement of kynurenic acid using isocratic reverse-phase high-performance liquid chromatography (HPLC) with fluorometric detection enhanced by Zn2+ as a postcolumn reagent. The method requires no prior sample preparation procedures other than extraction with 0.1 M HClO4. The reliability of the primary fluorometric method was verified by comparing measurements of tissue concentrations of kynurenic acid in human cerebral cortex and putamen using three different methods of separation with fluorometric detection, as well as four methods utilizing HPLC with coulometric electrode array system (CEAS) detection. All seven methods produced comparable results. The concentration of kynurenic acid in human cerebral cortex was 2.07 +/- 0.61 pmol/mg protein, and in human putamen, 3.38 +/- 0.81 pmol/mg protein. Kynurenic acid was also found to be present in human cerebrospinal fluid (CSF) at a concentration of 5.09 +/- 1.04 nM. The regional distribution of kynurenic acid in the rat brain was examined. Kynurenic acid concentrations were highest in brainstem (149.6 fmol/mg protein) and olfactory bulb (103.9 fmol/mg protein) and lowest in thalamus (26.0 fmol/mg protein). There were no significant postmortem changes in kynurenic acid concentrations in cerebral cortex, hippocampus, and striatum at intervals ranging from 0 to 24 h. Perfusion of the cerebral vasculature with normal saline prior to sacrifice did not significantly alter kynurenic acid content in rat hippocampus, cerebral cortex, or striatum. The analytical methods described are the most sensitive (10-30 fmol injection-1) and specific (utilizing both excitation and emissions properties and electrochemical reaction potentials, respectively) methods for determining kynurenic acid in brain tissue extracts and CSF. These methods should prove useful in examining whether kynurenic acid modulates EAA-mediated neurotransmission under physiologic conditions, as well as in determining the role of kynurenic acid in excitotoxic neuronal death.

Ion-selective microelectrodes suitable for recording rapid changes in extracellular ion concentration.

A method for fabricating double-barrel, ion-selective microelectrodes with fine tips (0.5-1.5 microns) and rapid response times is described. When made into K(+)-selective microelectrodes, the electrodes respond to changes in [K+]o with a time constant of 70-95 ms. The electrical response of these electrodes to common-mode voltages can be made to have a time constant of less than 2 ms, which minimizes electrical artifacts from field potentials. The application of these microelectrodes to the measurement of rapid, transient changes in retinal [K+]o is presented.

Estimation of extracellular volume in preterm infants less than 1500 g, children, and adults by sucrose dilution.

Extracellular volume can be estimated from the distribution volume of sucrose (Vdsucrose). The purpose of this study was to establish sucrose pharmacokinetics in preterm infants less than 1500 g compared to children and adults and to define an optimal sampling scheme. In five preterm infants, 10 children, and five adults Vdsucrose after a single injection was calculated with the two-compartment model (Vdsucrose-TCM) and with the one-compartment model applied only to the elimination phase of the same concentration-time curve (Vdsucrose-OCM). In preterm infants Vdsucrose-TCM was 417 +/- 45 mL/kg (mean +/- SD). Vdsucrose-OCM was only 3.0 +/- 2.3% higher, because sucrose elimination half-life was on average 250 times longer than distribution half-life. Therefore Vdsucrose-OCM, requiring only four blood samples between 2 to 5 h after injection, gave an adequate estimate of Vdsucrose in preterm infants less than 1500 g. Vdsucrose-TCM in children and adults was 188 +/- 26 and 189 +/- 17 mL/kg, respectively. Vdsucrose-OCM was 10 to 65% higher. Therefore, in children and adults only Vdsucrose-TCM gives a reliable estimate of Vdsucrose. This requires 10 to 15 blood samples. The reduced sampling scheme was used in an extension of the study of preterm infants including five additional infants. Vdsucrose-OCM in the preterm infants was 462 +/- 47 mL/kg at birth and 425 +/- 46 mL/kg at maximal postnatal wt loss. Postnatal wt loss (mean -83 +/- 44 g) was not significantly different from postnatal reduction of Vdsucrose-OCM (mean -82 +/- 56 mL), suggesting that postnatal wt loss mainly represents extracellular fluid loss.

In vivo analysis of extracellular proteins in rat brains with a newly developed intracerebral microdialysis probe.

Peptides and proteins in the extracellular space in the central nervous system were investigated in vivo using an intracerebral microdialysis probe. The molecular cut-off of the hollow fiber which was used for the probe was approximately 100 kDa. We examined recovery rates of several compounds in vitro. The recovery rates of proteins and peptides were between 7-28%, with the exceptions of substance P and insulin-like growth factor I. The recovery rates of monoamines and their metabolites were 22-40%. In in vivo studies, two major proteins with apparent molecular weights of 62 kDa and 12 kDa, and several minor proteins (28 kDa, 43 kDa, 52 kDa and 70 kDa) were detected by SDS-polyacrylamide gel electrophoresis in the dialysate from a probe implanted in the striatum of anesthetized rats. These results suggest that the newly developed, intracerebral microdialysis probe might be useful for investigating the dynamic changes of peptides and proteins in the central nervous system.

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.

Pulmonary capillary dynamics and fluid distribution after burn and inhalation injury.

A non-linear macroscopic mathematical model is described for the simulation of the different mechanisms that regulate the pulmonary capillary dynamics in patients with thermal injury. The techniques used in the construction of the model are those of 'system's dynamics'. This model has been incorporated into a patient simulator, which makes it possible to analyse the fluid and protein exchanges in a burn patient. The regulating mechanisms involved are: the pulmonary circulation, the fluid and protein transfer between the plasma and the interstitial space at the pulmonary capillary level, and the pulmonary lymphatic system. As a result of the sensitivity analyses of the model we propose that, for the simulation of the effects of an inhalation injury at the pulmonary capillary level, the parameters to be altered will be the pulmonary capillary permeability coefficient for proteins and the pulmonary capillary surface damaged by the injury. To verify the validity and utility of the model, the clinical progress of a series of burn patients with lung injuries has been compared with the results obtained using simulation.

Placental anticoagulant protein-I: measurement in extracellular fluids and cells of the hemostatic system.

Placental anticoagulant protein-I (PAP-I), a member of the lipocortin protein family, is a potent in vitro anticoagulant whose in vivo function is unknown. Very low levels of PAP-I were present in plasma of normal volunteers (0 to 5 ng/ml) and in randomly chosen plasma specimens from hospitalized patients (0 to 28 ng/ml). Review of selected hospital records did not reveal any single clinical entity that correlated with plasma levels. PAP-I was also found in amniotic fluid (12 to 107 ng/ml) and in conditioned medium of cultured endothelial cells (49 +/- 20 ng/ml). Gel filtration experiments showed that PAP-I was intact and uncomplexed in plasma and amniotic fluid. The protein was fairly abundant intracellularly: 4080 +/- 2560 ng/mg total protein in cultured umbilical vein endothelial cells; 178 +/- 109 ng/mg in platelets; 564 +/- 384 ng/mg in leukocytes; and 8.4 +/- 4.3 ng/mg in erythrocytes. The levels of PAP-I increased in platelet-rich plasma after stimulation of platelets with arachidonic acid but not after stimulation with ADP, epinephrine, thrombin, ristocetin, or collagen. These data suggest that PAP-I probably does not function as a circulating natural anticoagulant in normal persons.

Interstitial fluid concentrations of ceftriaxone (1 g.i.v.) in the subperitoneal space after hysterectomy.

The ability of an antibiotic to penetrate into the extravascular site of infection is particularly important for a successful perioperative antibiotic prophylaxis and postoperative therapy of bacterial infection. We, therefore, measured interstitial fluid concentrations of ceftriaxone in the subperitoneal space following hysterectomy using Rubinstein's disc method after intravenous administration of 1 g of ceftriaxone preoperatively. After removal of the uterus, two disc units were implanted intraoperatively in the right and left subperitoneal space of 16 patients and were drawn out through the open vaginal cuff after given periods of time. Five disc and blood specimens were obtained after 90 min and 2, 6, 12, 24, and 48 h, respectively. Ceftriaxone concentrations were determined by bioassay. After administration of 1 g of ceftriaxone, interstitial fluid concentrations following hysterectomy were above the MIC90 of most pathogens encountered in gynecologic infections over a period of 24 h.

Two-dimensional polyacrylamide gel electrophoresis of extracellular soybean pathogenesis-related proteins using PhastSystem.

Acidic and basic pathogenesis-related proteins (PR-Ps) were extracted from the intercellular fluid (IF) of soybean leaves, locally infected with tobacco necrosis virus and showing necrotic local lesions. Proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using PhastSystem and precast commercially available gels. Extracts from healthy leaves were run as controls. PR-Ps were first run under native PAGE conditions or isoelectric focusing (IEF), the gels stained with Coomassie Blue, then run under sodium dodecyl sulfate (SDS)-denaturing conditions and finally stained with silver. Ten major acidic PR-Ps were separated; their Mr's were close to those found by conventional PAGE. Their isoelectric points ranged from 3.5 to 5.0. Ten basic PR-Ps were separated and their Mr's estimated. None of these acidic or basic soybean PR-Ps was a glycoprotein. PAGE with PhastSystem and precast gels gives reliable results, comparable with those from conventional 2D-PAGE, with simpler experimental procedures. By electrophoresing Coomassie-stained gels with SDS in the second dimension, we were able to control the first-dimensional separation and to avoid laborious protocols generally adopted with unstained gels.