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Ultrastructural aspects of corneal fibrous tissue in the Scheie syndrome.

An ultrastructural study of the corneal fibrous tissue was performed in a case of Scheie's syndrome. Mucopolysaccharidosis deposits in keratocytes were observed as electron-clear and electron-dense inclusions. Modifications of the extracellular space included modifications of lamellar collagen organization and local hypertrophy of collagen bundles; presence of microfibrillar dense material isolating large irregular collagen fibers; and presence of fibrous long spacing type collagen fibers. The significance of these changes is discussed. This special form of collagen organization is supposed to appear in a modified microenvironment, that is the presence of an abnormal concentration of proteoglycans.

Biopsy↗

Nitric oxide acts directly in the presynaptic neuron to produce long-term potentiation in cultured hippocampal neurons.

Nitric oxide (NO) has been proposed to act as a retrograde messenger during long-term potentiation (LTP) in the CA1 region of hippocampus, but the inaccessibility of the presynaptic terminal has prevented a definitive test of this hypothesis. Because both sides of the synapse are accessible in cultured hippocampal neurons, we have used this preparation to investigate the role of NO. We examined LTP following intra- or extracellular application of an NO scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP.

Animals↗

Calcium mobilization and entry by thapsigargin in neuronal tumor cell line, SK-N-SH.

Using fura-2 loaded neural tumour cells, SK-N-SH, we demonstrate that receptor-mediated activation of phosphoinositide hydrolysis not only causes the release of Ca2+ from intracellular stores but also causes a concomitant influx of extracellular Ca2+. Thapsigargin (TG), a sesquiterpene lactone, causes a sustained elevation of intracellular Ca2+ and depletion of the inositol 1, 4, 5-trisphosphate-sensitive intracellular Ca2+ stores. In the absence of extracellular Ca2+, the increase in intracellular Ca2+ concentration ([Ca2+]i) was transient, suggesting that thapsigargin activates both intracellular mobilization and the influx of Ca2+ from extracellular space. These results are consistent with the proposal that the depletion of the inositol 1, 4, 5-trisphosphate-sensitive intracellular Ca2+ pool serves as a signal for Ca2+ influx.

Calcium↗

Metabolic response to two-stage adrenalectomy ih patients with Cushing's disease.

In 5 patients with Cushing's disease the balance of nitrogen, potassium, phosphorus, sodium and chlorides was determined during 5 days before, and 10 days after bilateral two-stage adrenalectomy. The patients received substitute treatment with hydrocortisone in mean doses of 112 mg/24 hours after the first stage and 203 mg/24 hours after the second stage. After removal of the first adrenal the metabolic response of the cell mass and extracellular space was within the normal range of moderately intense responses. After the second adrenalectomy the cumulative balance of the components of the cell mass was slightly negative and retention of sodium and chlorides was more pronounced than after the first operation.

Adolescent↗

[Kidney reaction to an increase in the volume of extracellular fluid in rabbits with ischemic cerebral hypertension].

The excretion of water, electrolytes and osmotically active substances and the blood plasma natriuretic activity were studied following isotonic dilation of the extracellular space in experiments on rabbits with ischemic cerebral hypertension. Rabbits with hypertension were marked by the phenomenon of exaggerated natriuresis and increased excretion of water and osmotically active substances after volumetric stimulation, which was due to a greater inhibition of processes of tubular reabsorption than in intact animals. The increase in the content of the natriuretic factor in the blood plasma of rabbits with ischemic cerebral hypertension after an increase in the volume of extracellular fluid could not be identified as one of the possible causes of inhibition of sodium reabsorption.

Animals↗

Transport, metabolism and distribution space of octanoate in the perfused rat liver.

The scope of the present work was to investigate the metabolism and the passage of octanoate from albumin into the phospholipid bilayer of the plasma membrane and from thence into the cell space. The experiments were done in the isolated perfused rat liver with infusions of albumin and octanoate at various concentrations. Once steady-state conditions were attained, trace amounts of [1-14C]-octanoate, [131 I]-albumin and [3H]-water were injected simultaneously and the effluent perfusate was fractionated. The normalized dilution curves were used for model analysis. The model which gives the best fit to the experimental results and which also produces the most consistent parameters is one that presupposes a rapid distribution of octanoate into the cell membrane and a slow transfer from the cell membrane into the cytosol. The concentration dependence of the distribution between the membrane and the extracellular space is parabolic, suggesting that octanoate changes the properties of the cell membrane when present at higher concentrations. The passage from the cell membrane into the cell space is relatively slow and limits metabolic transformation partly or totally, depending on the octanoate concentration in the plasma membrane. The rapid transfer of octanoate from the albumin space into the plasma membrane corroborates previous measurements of the dissociation of the albumin-octanoate complex.

Albumins↗

Blockers of carbonic anhydrase can cause increase of retinal capillary diameter, decrease of extracellular and increase of intracellular pH in rat retinal organ culture.

BACKGROUND: At least in normal-pressure glaucoma a vascular genesis with hypoperfusion and regulation impairment is discussed. This may lead to malnutrition of retinal ganglion cells and apoptosis. The retinal microvasculature has a small functional reserve. In addition, the retinal microvessels lack the autonomic nerves that are normally found in other tissues. Thus, no systemic influences reach the retinal capillaries apart from circulating hormones or transmitters. Blockers of carbonic anhydrase (CA) may modulate regional blood flow by mediating changes in extra- and intracellular pH. However, it is still unclear (1) whether blockers of CA really change the pH near the retinal capillaries and (2) how changes in the local pH affect the capillary tone in situ. Therefore, we tested dorzolamide and acetazolamide in our model of the freshly enucleated rat retina. METHODS: Adult Sprague-Dawley rats (of both sexes, 250-350 g) were killed and retinae were prepared. The retinae were gently separated from the retinal pigmented epithelium and were observed in a chamber for electronic light microscopy or were fixed for immunohistochemistry. Electronic light microscopy of the retinal cells was performed with a Zeiss Axiovert microscope equipped with differential interference contrast (DIC) optics. Changes in capillary diameter were measured using an Openlab acquisition system and analyzed statistically using ANOVA. In addition to light microscopy the intracellular pH was analyzed in the whole mounts by ratio imaging of the pH using the special dye BCECF-AM (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluoresceinacetoxymethyl ester) and the extracellular pH using BCECF (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein). RESULTS: Pericytes of most segments of retinal capillaries are immunoreactive for alpha-smooth muscle actin (SMA). The SMA immunostaining is strong around the nucleus; the endothelial tube is visible by virtue of the slight immunoreactivity of the surrounding pericyte processes. Acetazolamide and dorzolamide showed statistically significant vasoactive effects in retinal capillaries. Vasodilation increased by up to 105% of that in control capillaries after 5, 10 and 15 min. CA inhibitors were found to be able to induce intracellular alkalization in retinal cells. After addition of dorzolamide or acetazolamide the extracellular pH decreased from 7.4 to 7.2 concomitant with diameter changes. CONCLUSIONS: The tube-like pattern of SMA immunoreactivity demonstrates the presence of contractile elements within the pericyte processes of the rat retina. Thus, pericytes may act as a regulation element within the retinal microcirculation. Our results further suggest that CA inhibitors are able to decrease pH in the extracellular space; however, the pH within the cells increases. The increase in capillary diameter is concomitant with these pH changes. Thus, we may conclude that CA inhibitors can relax pericytes and might improve the retinal blood supply.

Acetazolamide↗

Transepithelial permeability in the rabbit pancreas.

1. The transepithelial permeability in the isolated rabbit pancreas has been studied with the aid of radioactive markers added to the bathing medium. 2. After addition of these compounds in 2 mM concentration to the medium, they equilibrate within 30 min to a steady-state concentration in the secreted fluid. The latter concentrations, expressed as percent of those in the bathing medium, are: urea 100%, glycerol 90%, erythritol 95%, mannitol 60%, lactose 5%, sucrose 4% and inulin 3%. 3. Addition of 10(-5) M carbachol to the bathing medium after 60 or 90 min of incubation results in an increase of the concentrations of mannitol, lactose sucrose and inulin in the secreted fluid. Maximal concentrations, reached about 35 min after addition of the stimulant, are: mannitol 65%, lactose 31%, sucrose 23%, inulin 8%. 4. No change in the concentration of urea is observed, while the concentrations of glycerol and erythritol increase always to 100% after addition of 10(-5) M carbachol. 5. For sucrose and lactose the increase in permeability appears to be dependent on the concentration of carbachol. 6. There is no increase in the extracellular space for lactose, sucrose and inulin after incubating fragments of the rabbit pancreas with 10(-5) M carbachol. 7. Addition of atropine 5 min or more after carbachol stimulation has no effect on enzyme secretion, but markedly inhibits the increase in sucrose permeability. 8. These results indicate that: (a) the permeability of the transcellular transport route in the isolated rabbit pancreas is determined by the size of the permeating molecules, (b) this route is probably extracellular, (c) its permeability is increased by a cholinergic agent in dose-dependent fashion, (d) the increase in permeability is not caused by the enzyme secretion as such.

Animals↗

Uptake of labelled noradrenaline by isolated atria.

The uptake of labelled noradrenaline by isolated rabbit atria has been studied, and the rate of outflow of radioactivity after a period of loading has been recorded. There is first a rapid outflow presumably from extracellular space, followed by a slow outflow presumably from intracellular space. Cocaine greatly diminished the intracellular uptake. Tyramine caused an increased outflow of radioactivity from intracellular sites which was not due to the increase in atrial rate, since noradrenaline which increased the rate more than tyramine had less effect on the outflow.

Animals↗

Simultaneous monitoring of electrochemical and unitary neuronal activities by a single carbon fiber microelectrode.

Simultaneous measurements of electrochemical and electrophysiological changes in the substantia nigra pars compacta of the rat were tried with a single carbon fiber microelectrode. Electrochemical detection of catecholamines was done by differential pulse voltammetry. The effects of haloperidol on the level of catecholamines in extracellular spaces and on dopaminergic neuronal discharges were investigated. Haloperidol induced an increase in unitary discharges parallel to the elevation of the catecholamine level. With this technique, direct information can be obtained on the relationship between released catecholamines and unitary neuronal activity.

Animals↗

Growth hormone attenuates the abnormal distribution of body water in critically ill surgical patients.

BACKGROUND: Catabolic illness is associated with fluid retention and extracellular space expansion. To determine the effect of human growth hormone (GH) on body water compartments, critically ill surgical patients were studied for a 2-week period during which they either continued to receive standard intensive care unit support, or in addition, received GH, 10 mg/day. METHODS: Body water compartments were measured at the beginning and end of the period by the indicator dilution technique with sodium bromide and heavy water used as the indicators of extracellular (ECW) and total body water (TBW), respectively; intracellular water (ICW) was calculated by subtraction. RESULTS: Neither group lost significant amounts of weight or TBW. A marked ECW expansion and disturbance of the ECW/TBW ratio occurred in the patients receiving standard care, which was associated with a dramatic reduction in ICW, a critical component of the body cell mass (BCM). In contrast, GH-treated patients maintained ECW and ICW, indicating a preservation of BCM, and their ECW/TBW ratio normalized. CONCLUSIONS: GH administration prevents ECW retention and stabilizes or normalizes fluid distribution during critical illness. Taken together with its known anabolic effects under these conditions, the maintenance of ICW demonstrates that GH can be used to preserve BCM in complex surgical patients.

Body Water↗

Diffusion of radiotracers in normal and ischemic brain slices.

Diffusion in the extracellular space (ECS) is important in physiologic and pathologic brain processes but remains poorly understood. To learn more about factors influencing tissue diffusion and the role of diffusion in solute-tissue interactions, particularly during cerebral ischemia, we have studied the kinetics of several radiotracers in control and hypoxic 450-microm hippocampal slices and in 1,050-microm thick slices that model the ischemic penumbra. Kinetics were analyzed by nonlinear least squares methods using models that combine extracellular diffusion with tissue compartments in series or in parallel. Studies with 14C-polyethylene glycol confirmed prior measurements of extracellular volume and that ECS shrinks during ischemia. Separating diffusion from transport also revealed large amounts of 45Ca that bind to or enter brain as well as demonstrating a small, irreversibly bound compartment during ischemia. The rapidity of 3H2O entry into cells made it impossible for us to distinguish intracellular from extracellular diffusion. The diffusion-compartment analysis of 3-O-methylglucose data appears to indicate that 5 mmol/L glucose is inadequate to support glycolysis fully in thick slices. Unexpectedly, the diffusion coefficient for all four tracers rose in thick slices compared with thin slices, suggesting that ECS becomes less tortuous in the penumbra.

Animals↗

The pathology of experimental obstructive hydrocephalus. Electron microscopic observations.

Obstructive hydrocephlus was produced in 10-14 day-old rabbits by injection of kaolin into the cisterna magna and the ependyma and subependymal tissue was studied by electron microscopy. Generally, the study confirmed recent light microscopic observations on similar models (Torvik et al., 1976). In contrast to most previous reports, it was found that the ependyma adapted remarkably well to ventricular dilatation. No true ependymal defects occurred even in extensive hydrocephalus except at the sites of the ventricular synechiae which sometimes ruptured. The specialized ependymal junctions remained intact but outside the junctions the intercellular clefts were widened, particularly along the lateral wall of the lateral ventricle. The density of the microvilli and cilia decreased, probably because of the increase in the surface area of the ependyma. Dense bundles of filaments developed in the ependymal cells of the hydrocephalic animals. The extracellular space of the subependymal white matter appeared increased but there was no evidence of destruction of fibres or cells. Thus, the reduction of the cerebral mantle thickness was probably mainly caused by pre-sure atrophy.

Animals↗

Swelling of rat mesangial cells induces a Ca2+-dependent Cl- conductance.

Membrane voltage (Vm) and ion currents of rat mesangial cells in primary culture were measured with the patch-clamp technique in the fast whole-cell configuration. Vm was -44 +/- 1 mV (n = 138). A reduction of the osmolality from 290 to 190 mosmol/kg depolarized Vm from -44 +/- 1 to -29 +/- 1 mV (n = 118) and increased the inward and outward conductances (Gm) from 14 +/- 2 to 39 +/- 4 nS and 13 +/- 2 to 37 +/- 4 nS (n = 84), respectively. During the hypotonicity-induced depolarization the cell capacitance increased significantly from 33 +/- 3 to 42 +/- 4 pF (n = 40). The effect of hypotonic cell swelling on Vm was increased in a bath with a reduced extracellular Cl- of 32 mmol/l (by 71 +/- 4%, n = 23), indicating that a Cl- conductance was activated. The permselectivity of this conductance was I- > or = Br- > Cl-. The Vm response was not affected in the presence of a reduced extracellular Na+ of 5 mmol/l (n = 13) and was inhibited in a solution with reduced extracellular Ca2+ concentration (by 63 +/- 9%, n = 14). In microfluorescence measurements with the Ca2+-sensitive dye fura-2 hypotonic cell swelling induced a sustained increase of the intracellular Ca2+ activity, [Ca2+]i (n = 19). The increase of [Ca2+]i was completely inhibited when the extracellular solution was free of Ca2+. The Vm response to hypotonic cell swelling was not attenuated in the presence of the L-type Ca2+ channel blockers nicardipine (n = 5), nifedipine (n = 5) and verapamil (n = 5) (all at 1 micromol/l). The data indicate that in rat mesangial cells, osmotic swelling induces a Ca2+ influx from extracellular space. This Ca2+ influx activates a Cl- conductance resulting in a depolarization of Vm. The enhanced Cl- conductance may lead to KCl extrusion and hence regulatory volume decrease.

Animals↗

Freezing avoidance and the distribution of antifreeze glycopeptides in body fluids and tissues of Antarctic fish.

The distribution of antifreeze glycopeptides (AFGPs) in the body fluids and tissues of antarctic notothenioid fish was determined. In Dissostichus mawsoni (Norman), the peritoneal, pericardial and extradural fluid, like the blood, contained all eight AFGPs and in concentrations sufficient to depress freezing points below that of sea water (-1.9 degree C). Secreted fluids including urine, endolymph and aqueous and vitreous humour either lack all AFGPs or have very low concentrations of only the low molecular weight forms and have freezing points of about -1.0 degree C, and are therefore undercooled with respect to environmental temperature. Fluids with high concentrations of AFGPs also contain high levels of proteins similar to plasma proteins. Systemic administration of tritiated AFGPs in the closely related species Trematomus bernacchii (Boulenger) yielded a distribution pattern similar to that of the native AFGPs in D. mawsoni. This suggests passive distribution of AFGPs into the various fluid compartments following secretion from the liver; a pattern typical of secreted blood proteins. Tissue distribution of AFGPs was determined by comparison with that of the extracellular space marker [14C]polyethylene glycol. AFGPs were found in the interstitial fluid of all body tissues examined except brain tissue. No tissue showed any intracellular accumulation of tritiated AFGPs from the blood.

Animals↗

Body fluids and their distribution in experimental hypertension.

The relation between blood pressure level and extracellular fluid volume and its distribution was studied in rats subjected to the following hypertensive stimuli--1K1C and 2K1C renal artery constriction, subtotal nephrectomy-salt and DOCA-salt. In all experimental groups the blood pressure increase was accompanied by increased extracellular fluid volume which was not always distributed proportionally between intravascular (PV) and interstitial (IFV) compartments. The blood pressure rise was further potentiated by plasma volume expansion so that the increased PV/IFV ratio was associated with a more pronounced hypertensive response (1K1C vs 2K1C, DOCA-salt vs subtotal nephrectomy-salt). However, adequate expansion of interstitial fluid is a necessary prerequisite for the hypertensive response. In DOCA-salt treated DI Brattleboro rats (lacking antidiuretic vasopressin action) plasma volume expansion per se was not accompanied by severe DOCA-salt hypertension. It is concluded that the expansion of both compartments of extracellular space, i.e. plasma volume and interstitial fluid volume, was necessary for a full development of severe hypertension. The expansion of only one of these compartments was accompanied by a mild blood pressure increase or blood pressure did not change significantly.

Animals↗

Alterations in hepatocytes of mice fed a gallstone-inducing diet: occurrence of nuclear and cytoplasmic lipids.

Mice fed a high cholesterol-cholic acid (lithogenic) diet for one year develop fatty livers in addition to gallstones. Light and electron micrographs demonstrate large amounts of lipids in liver parenchymal cells, often to the exclusion of most other cytoplasmic organelles. In addition, some hepatocytes exhibit nuclear lipid pseudo- and true inclusions. Other prominent features of hepatocytes after lithogenic diet include segregation of nucleolar granular and fibrillar material. Accumulation of considerable collagen in extracellular spaces is also noted. Observations suggest changes induced by the cholesterol diet are comparable to cytologic alterations seen in spontaneous and drug induced hepatic tumors, as well as to more general "fatty metamorphosis" of the liver.

Animals↗

Ultrastructural changes in the neurohypophysis of the aged male rat.

The neurohypophyses (neural lobes of the pituitary) of young adult (3-6 months) and aged (12-30 months) male rats were studied by means of electron microscopy. Stereomorphometric analyses were performed to determine the size, number and relative volume of the hormone-containing vesicles. The principal observations included: 1) the conspicuous depletion in aged rats of the granular hormone-containing vesicles from the axon terminals and the Herring bodies, with a decrease in the relative volume fraction of the vesicles from 4.8 Vv% in the control animals to 1.1 Vv% in the aged rats; 2) a change in the size-distribution of the hormone vesicles; 3) an increase in the extracellular space around the nerve terminals, axons and capillaries; and 4) lipid accumulations and signs of activation in the pituicytes. The possible physiological significance of the findings is discussed in the light of several regulatory functions known to be altered during the process of ageing.

Aging↗