Space Marker Research Articles

Calcium flux measurements during hypoxia in cultured heart cells

We tested the hypothesis that cultured chick embryo ventricular cells grown in monolayer could be used to study calcium fluxes across the myocardial sarcolemmal membrane during simulated ischemia. A specially adapted anaerobic chamber allowed the exposure of heart cells to profound hypoxia (PO2 less than 1.5 Torr) for prolonged periods of time. Ca2+ flux studies were conducted in this chamber following hypoxia and substrate deprivation to simulate important components of myocardial ischemia. To prove we were measuring cellular rather than interstitial cation contents we conducted 51Cr-EDTA interstitial space marker washout studies and showed that our procedures were sufficient to remove at least 99.7% of the extracellular fluid. The addition of lanthanum (1 mM) to the wash solution reduced nonspecific 45Ca2+ binding to the polystyrene culture plates to less than 0.03% of applied counts, but did not alter 45Ca2+ uptake under normoxic conditions. Following 2 h of hypoxia and substrate deprivation the Ca2+ content of the rapidly exchangeable Ca2+ pool of cultured monocytes increased 282% compared to control values during normoxia. We conclude that cultured chick embryo ventricular cells grown in monolayer are suitable for investigation of cation fluxes during simulated ischemia. Under the conditions studied, lanthanum displaceable Ca2+ did not make a major contribution to Ca2+ influx. The system permitted clear resolution of alterations in Ca2+ flux kinetics under conditions of profound hypoxia.

Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout ( Salmo gairdneri)

1. 1. Total blood volume and relative blood volumes in selected tissues were determined in non-anesthetized, confined rainbow trout by using 51Cr-labelled trout erythrocytes as a vascular space marker. 2. 2. Mean total blood volume was estimated to be 4.09 ± 0.55 ml/100 g, or about 75% of that estimated with the commonly used plasma space marker Evans blue dye. 3. 3. Relative tissue blood volumes were greatest in highly perfused tissues such as kidney, gills, brain and liver and least in mosaic muscle. 4. 4. Estimates of tissue vascular spaces, made using radiolabelled erythrocytes, were only 25–50% of those based on plasma space markers. 5. 5. The consistently smaller vascular volumes obtained with labelled erythrocytes could be explained by assuming that commonly used plasma space markers diffuse from the vascular compartment.

Influence of Saponins on Gut Permeability and Active Nutrient Transport in Vitro

The influence of four saponins, three triterpenoid glycosides and one steroidal amine glycoside, upon intestinal transport was investigated in vitro. In the presence of Gypsophylla saponin, carrier-mediated galactose transport was inhibited, although the uptake of the passively transported L-isomer of glucose increased. The uptake of the extracellular space marker, polyethylene glycol 4000, was also higher, indicating that the saponin inhibited active transport by increasing the general permeability of the enterocytes. Gypsophylla saponin, in contact only with the mucosal surface of everted jejunal sacs, induced a rapid decline in glucose-stimulated transmural potential difference. The rate of decline increased as the saponin concentration was raised over the approximate range of 0.3 to 8 mM. Saponaria saponin and α-tomatine also reduced transmural potential difference, but soya saponins were much less effective. The results indicate that some saponins readily increase the permeability of the small intestinal mucosal cells, thereby inhibiting active nutrient transport, and facilitating the uptake of materials to which the gut would normally be impermeable.

Effect of CO 2 on a brain extracellular space marker and evidence of its neuronal modulation

Increases in inspired CO 2 consistently altered the local concentration of the brain extracellular space marker α-naphthalene sulfonate (α-NS) as measured with ion-selective micropipettes in the rat thalamus. Stereotaxic injection of lidocaine in the region of the loccus coeruleus attenuated this effect of CO 2, and amittriptyline, a tricyclic anti-depressant and amine reuptake inhibitor, potentiated the effect. These results suggest that metabolic demand, as mimicked here by the addition of CO 2, alters the fluid environment of the brain and central noradrenergic mechanisms may modulate this response.

Lymphatic Transport of Liposome-Encapsulated Agents: Effects of Liposome Size Following Intraperitoneal Administration

Liposomal encapsulation can limit passage of a drug from a peritoneal administration site to blood, while enhancing lymphatic transport. We evaluated the effects of liposome size on lymphatic transport after intraperitoneal administration. Liposomes tested had mean diameters of either 0.72, 0.46, 0.17, or 0.048 μm and identical compositions. [14C]Sucrose was the aqueous space marker (model drug). Doses were given to thoracic-duct-cannulated rats. The subsequent 0–5h time-course of carbon-14 was quantified in thoracic lymph, several lymph nodes, blood, and urine. Calibration studies indicated a maximum of ∼30% of the absorbed dose could be collected in thoracic lymph. Carbon-14 levels in the various nodes covered a 1000-fold range, and relatively high levels were observed in the left mediastinal, parathymic, cisternal, and renal lymph nodes. Liposome stability in vivo and in vitro increased with decreasing size. Absorption from the peritoneal cavity was independent of size. The smallest liposomes were collected in lymph with little lymph node retention. The largest liposomes were retained most by lymph nodes, and would be the best prototypical carrier of the group if increased therapeutic availability within both lymph and lymph nodes is desired. The results implicate other, unexplored physical and physiological variables as potentially of equal importance.

Alpha-adrenoceptor stimulation, lysophosphoglycerides, and lipid peroxidation in reoxygenation induced calcium uptake in rabbit myocardium.

Calcium uptake on reoxygenation of hypoxic cardiac muscle is well documented. Alpha-adrenoceptor stimulation by released catecholamines, lysophosphoglycerides and lipid peroxidation have all been suggested as mediators of this effect. We have measured the uptake of Ca2+ on reoxygenation in the isolated arterially perfused interventricular septum of the rabbit heart. The alpha agonist phenylephrine (1 mumol X litre-1) did not alter calcium uptake, and the presence of either prazosin (1 mumol X litre-1) or phentolamine (10 mumol X litre-1) did not alter the reoxygenation induced Ca2+ uptake. Lysophosphatidylcholine caused an increase in Ca2+ uptake above 8 mumol X litre-1 but also produced a simultaneous increase in the distribution volume of 51Cr-EDTA, an extracellular space marker, indicating loss of membrane integrity. Hydrogen peroxide and cumene hydroperoxide both caused an increased Ca2+ uptake, but no disruption of the cell membrane; the effect on Ca2+ uptake could be inhibited by Ni2+ ions. Alpha adrenergic stimulation and lysophosphoglycerides do not appear to be key to Ca2+ uptake on reoxygenation, but lipid peroxidation of the sarcolemma is a possible mechanism.

Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides.

Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4°C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161–176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca 2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4°C and 37°C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.

Differential permeability of uterine and liver vascular beds to estrogens and estrogen conjugates.

The role of capillary membrane permeability and the effect of plasma protein binding on the influx of unconjugated and conjugated estrogens into a target organ, the uterus, and a metabolic organ, the liver, were studied in anesthetized rats. In the absence of plasma proteins, estrone (E1) and estradiol (E2) were freely diffusible through the uterine capillaries, but influx was significantly reduced for estriol (E3) and estetrol. In the uterus, the influx of the conjugated estrogens was markedly restricted and approximated the influx of dextran, a vascular space marker. The polarity of the compound (based on the number of hydrogen bond-forming functional groups and the presence of charged groups) appeared to predict uterine endothelial membrane permeability better than the octanol/Ringer's partition coefficient. In contrast to the selective permeability properties of the uterine endothelial barrier, the limiting membrane lining the hepatic microcirculation, the hepatocyte cell membrane, was highly permeable to all unconjugated and conjugated estrogens. The addition of 4% albumin to the injection solution led to a significant inhibition of uterine influx of E2, but not E1 or E3. In the liver, only the influx of E1 sulfate was slightly diminished by 4% albumin. In all cases, the influx of estrogens greatly exceeded the rate that would be expected if only the fraction that was free (dialyzable) in vitro was diffusible in vivo. Human sera containing sex hormone-binding globulin and albumin caused inhibition of influx of E1 and E2 through the uterine capillary barriers, whereas in the liver, the influx of E2 sulfate, and E3 glucuronide were diminished. The results are compatible with a difference in permeability of the microvasculature of the two organs and a differential availability of protein-bound estrogen for influx into liver and uterus. With the exception of E1, which is nearly completely diffusible into both organs, the influx of estrogens and estrogen conjugates into liver is greatly amplified compared to that into a peripheral organ such as the uterus.

Flow cytofluorometric investigation of the uptake by hepatocytes and spleen cells of targeted and untargeted liposomes injected intravenously into mice

Untargeted liposomes (composition: PC-PS-cholesterol) and targeted liposomes (composition: PC-PS-cholesterol-lactosylceramide) having encapsulated concentration-quenched carboxyfluorescein were injected intravenously into mice. 1 h after injection, the mice livers were perfused, excised and the hepatocytes were separated from nonparenchymal cells and analysed in a fluorescence-activated cell sorter analyzer. The result was that hepatocytes took up significantly more liposomes when lactosylceramide was inserted in the liposome bilayers, which was in good agreement with observations made on the in vivo uptake of liposome-encapsulated insulin gene (Soriano, P. et al. (1983) Proc. Natl. Acad. Sci. USA, 80, 7128–7133). Cytofluorimetric analysis of the spleen cells showed that approx. 10% of the splenic lymphocytes take up high amounts of lactosylceramide liposomes, whereas most of the phospholipid liposomes are taken up by the phagocytic cells. The flow cytofluorimetric analysis shows, moreover, the internalization of the liposomes by the target cells and allows a quantitation of this uptake. Thus, in vivo targeting of the liposomes to specific liver and splenic cells, by means of glycolipid insertion in the liposome bilayer, is shown to take place with delivery of the liposomal aqueous space marker to these cells.

Characterization of liposomes prepared using a microemulsifier

A new type of device can prepare liposomes continuously, in large quantities and with excellent aqueous space capture efficiency. At initial lipid concentration of 300 μmol/ml these liposomes capture approx. 75% of cytosine arabinoside used as an aqueous space marker. Liposome size can be reduced by increasing the number of times the preparations are recycled through the microemulsifier. Liposomes less than 0.1 μm in diameter, as shown by electron microscopy, can be made easily. Liposomes prepared at 300 μmol/ml, composed of phosphatidylglycerol/phosphatidylcholine/cholesterol in a 0.1:0.4:0.5 molar ratio leaked less than 1% of entrapped cytosine arabinoside (Ara-C) at 4°C, and less than 10% Ara-C at 37°C plus serum, over a 48 h period. These liposomes could be useful for a number of applications including diagnostics, therapeutics and model membrane studies.

Intrahepatic uptake and processing of intravenously injected small unilamellar phospholipid vesicles in rats

Small unilamellar vesicles consisting of sphingomyelin, cholesterol and phosphatidylserine in a molar ratio of 4:5:1 containing [ 3H]inulin as a marker of the aqueous space or [ Me- 14C]choline-labeled sphingomyelin as a marker of the lipid phase were injected intravenously into rats. After separation of the non-parenchymal cells into a Kupffer cell fraction and an endothelial cell fraction by elutriation centrifugation analysis of the radioactivity contents demonstrated that Kupffer cells were actively involved in the uptake of the vesicles whereas endothelial cells did not contribute at all. Uptake by total parenchymal cells was also substantial but, on a per cell base, significantly lower than that by the Kupffer cells. By comparising the fate of the [ 3H]inulin label and the [ 14C]sphingomyelin label it was concluded that release of liposomal lipid degradation products especially occurred from Kupffer cells rather than from parenchymal cells. In both cell types, however, substantial proportions of the 14C-label accumulated in the phosphatidylcholine fraction, indicating intracellular degradation of sphingomyelin and subsequent phosphatidylcholine synthesis. Treatment of the animals with the lysosomotropic agent chloroquine prior to liposome injection effectively blocked the conversion of the choline-labeled sphingomyelin into phosphatidylcholine in both cell types. This observation indicates that uptake of the vesicles occurred by way of an endocytic mechanism.

A stopped flow capillary perfusion method to evaluate contraluminal transport parameters of methylsuccinate from interstitium into renal proximal tubular cells.

In order to study the transport of dicarboxylic acids through the contraluminal cell membrane of proximal tubular cells, 3H- methylsuccinate has been synthetized by catalytic hydration of methylfumarate . As the chromatography of radioactive material excreted in the urine after i.v. injection of 3H- methylsuccinate shows, no metabolite is detectable during the first 3 min. After 10 min, less than 10% of the excreted radiolabel is metabolized. To measure the contraluminal influx of 3H- methylsuccinate from the interstitium into cortical tubular cells, the renal vessels were clamped so that the proximal tubular lumina collapsed. Then Ringer solution was injected into the blood capillaries. It contained different concentrations of 3H- methylsuccinate and 14C-inulin as extracellular space marker. After contact times between 1 and 10 s, this fluid was withdrawn from the capillaries and the disappearance of 3H- methylsuccinate relative to 14C-inulin was measured. The morphological compartments in the outer cortex of the clamped glutaraldehyde-fixed kidney were evaluated by a stereological method. For proximal tubular cells a ratio of extracellular water space to intracellular space of 1:3.1 and a ratio extracellular water space to free cell water space of 1:2 was found. It was tested whether the experimental disappearance curves with 4 different starting concentrations of 3H- methylsuccinate fit with the data from four model calculations. It was found that the data and the conditions of transport are consistent with the predictions of a facilitated diffusion model.(ABSTRACT TRUNCATED AT 250 WORDS)

99mTc-labeled nucleotides as tumor-seeking radiodiagnostic agents.

Several lines of human tumor cells in monolayer and soft agar cultures allow permeation of low levels of adenine nucleotides through their plasma membranes, while, in general, untransformed cells do not incorporate adenine nucleotides into their cellular pools without prior degradation of the nucleotides to adenosine. This study determined the uptake of 99mTc-radiolabeled chelated forms of adenine nucleotides, 99mTc-Ap4A (diadenosine 5',5"',P1,P4-tetraphosphate) and 99mTc-ATP chelates as radiodiagnostic agents suitable for the in vivo detection of tumors by radionuclide imaging. Biodistribution studies revealed that Ap4A accumulated preferentially in RT-24 tumors implanted in rats and that V2 carcinoma implanted in rabbits could be readily visualized by in vivo imaging. The biodistribution at various time points showed increased tumor-to-muscle ratios after 99mTc-Ap4A or 99mTc-ATP injections when compared with a nonspecific marker of the extracellular fluid space, 99mTc-labeled diethylenetriaminepentaacetic acid and with an agent known to localize in some tumors, 67Ga-labeled citrate. Studies of ectoenzymatic activities of virus-transformed animal cells and their untransformed counterparts in monolayer cultures showed marked decreases in the ectoenzymatic activities that degrade Ap4A in the transformed cells. Incorporation of en bloc [3H, 32P]Ap4A into cellular acid-soluble nucleotide pools of certain transformed cells was observed. Normal untransformed cells incorporated the radioactive label only by prior degradation to [3H]adenosine and 32Pi.

Creatine phosphate: An additive myocardial protective and antiarrhythmic agent in cardioplegia

The potential for enhancing myocardial protection by adding high-energy phosphates to cardioplegic solutions was investigated in a rat heart model of cardiopulmonary bypass and ischemic arrest. Creatine phosphate (CP) was evaluated as an additive to the St. Thomas' Hospital cardioplegic solution. Dose-response studies (CP 0 to 50 mmol/L) revealed 10.0 mmol/L as the optimal concentration which improved recovery of aortic flow and cardiac output after a 40 minute period of normothermic (37 degrees C) ischemic arrest from 21.2% +/- 5.4% and 32.8% +/- 4.6% in the CP-free control group to 82.5% +/- 3.7% and 82.6% +/- 4.2% (p less than 0.001), respectively. Creatine kinase (CK) leakage was reduced by 68.7% (p less than 0.001) in the CP group. With hypothermic (20 degrees C) ischemia (240 minutes) and multidose (every 30 minutes) cardioplegia, recoveries of aortic flow and cardiac output were improved from 33.1% +/- 8.4% and 42.2% +/- 7.7% in the CP-free control group to 77.9% +/- 4.2% and 79.6% +/- 4.3% (p less than 0.001), respectively, in the drug group. In addition to improving function and decreasing CK release, CP reduced reperfusion arrhythmias, significantly decreasing the time between cross-clamp removal and return of regular rhythm and also completely obviating the need for electrical defibrillation. 51Chromium-ethylenediaminetetraacetic acid (51Cr-EDTA), an extracellular space marker, was used to study the disappearance of CP from the cardioplegic solution during its stasis in the heart. Upon reperfusion, two thirds of the infused dose appeared unchanged in the coronary effluent; the remainder was either degraded or accumulated by the myocardium. Despite its alleged inability to enter the myocardial cell, exogenous CP exerts potent protective and antiarrhythmic effects when added to the St. Thomas' Hospital cardioplegic solution. Although the mechanism of action remains to be elucidated, it may involve binding or uptake of the drug.

Liposome-mediated transfer of macromolecules into flagellated cell envelopes from bacteria.

We have studied the interaction between flagellated cell envelopes from Escherichia coli and liposomes. Oligolamellar liposomes of ca. 0.45-micron diameter, composed of azolectin, phosphatidylserine, and cholesterol at a molar ratio of 7:1:2, were prepared by freezing and thawing and subsequent extrusion through polycarbonate filters. These liposomes exhibited high entrapment capacity and low leakiness. Liposome-cell envelope interaction was monitored flow cytometrically in a fluorescence-activated cell sorter with a fluorescent aqueous space marker and by a filtration assay with radiolabels for the lipid phase and the liposomal aqueous space. Maximal association of liposomes with the envelopes was observed in both assays after ca. 25 min at 30 degrees C. After such period of time, it seems that up to 200 liposomes (depending on the liposome to envelope ratio) were associated with a single cell envelope, as calculated from the radiotracer studies. Fluorometric measurements of the transfer of liposomal contents and the intermixing of membrane lipids indicated that at least 20% of the envelope-associated liposomes had delivered their content into the envelopes, possibly by fusion. Electron microscopic observations confirmed the transfer of liposome-encapsulated ferritin molecules into the cell envelopes. Our data suggest that liposomal carriers might be employed to deliver cytoplasmic, chemotaxis-related macromolecules into bacterial cell envelopes.

Interaction of liposomes with Kupffer cells in vitro

We investigated the interaction of liposomes with rat Kupffer cells in monolayer maintenance culture. The liposomes (large unilamellar vesicles, LUV) were composed of 14 C-labelled phosphatidylcholine, cholesterol and phosphatidylserine (molar ratio 4 : 5 : 1) and contained either 3 H-labelled inulin or 125 I-labelled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. After 2–3 days in culture the cells exhibited optimal uptake capacity. The uptake process showed saturation kinetics, maximal uptake values amounting to 2 nmol of total liposomal lipid/h/10 6 cells. This is equivalent to 1500 vesicles per cell. The presence of fetal calf serum (FCS) during incubation increased uptake nearly two-fold, whereas freshly isolated rat serum had no effect. The binding of the liposomes to the cells caused partial release of liposomal contents (about 15–20%) both at 4°C and at 37°C. In the presence of metabolic inhibitors the uptake at 37°C was reduced to about 20% of the control values. Inulin and lipid label became cell-associated at similar rates and extents, whereas the association of albumin label gradually decreased after attaining a maximum at relatively low values. When, after 1 h incubation, the liposomes were removed continued incubation for another 2 h in absence of liposomes led to an approx. 30% release of cell-associated lipid label into the medium in water-soluble form. Under identical conditions as much as 90% of the cell-associated albumin label was released in acid-soluble form. Contrarily, the inulin label remained firmly cell-associated under these conditions. From these results we conclude that Kupffer cells in monolayer culture take up liposomes primarily by way of an adsorptive endocytic mechanism. This conclusion was confirmed by morphological observations on cells incubated with liposomes containing fluorescein isothiocyanate (FITC) dextran or horseradish peroxidase as markers for fluorescence microscopy and electron microscopy, respectively.

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).

Relation between the increase in the diffusional permeability of the blood-central nervous system barrier and other changes during the development of experimental allergic encephalomyelitis in the lewis rat

The reduction in the effectiveness of the blood-brain and blood-spinal cord barriers, previously seen in rats at the height of the acute episode of experimental allergic encephalomyelitis, has now been measured at various stages in the development of the disease up to 60 days after inoculation with guinea pig spinal cord in complete Freund's adjuvants. The marker of extracellular space, radioactively labelled mannitol, only crosses the blood-central nervous system barriers very slowly by passive diffusion in normal rats. An abnormal penetration of this marker into the central nervous system began to develop during the second week after inoculation, appearing first in the lower spinal cord, where it also reaches the highest level during the acute phase of the attack. The leak begins before either the clinical signs become evident or cuffing is seen around blood vessels in stained sections. As the clinical signs are disappearing, from about 15 days onwards, the permeability of the barrier returns steadily to its normal low value, starting in the spinal cord, especially the caudal part. The timing of the reduction in the effectiveness of the blood-central nervous system barrier in relation to other clinical and histological changes suggests that it may play a part in the development of the lesion. The relation between the timing of these changes in EAE and that in the development of a new lesion in (exacerbation of) multiple sclerosis is discussed.

LIGHT SENSITIVE LIPOSOMES

AbstractTwo phospholipids were designed and synthesized that have the property of forming liposomes which undergo a dramatic increase in permeability when irradiated with light (360 nm). Liposomes prepared from these phospholipids leak 100% of entrapped aqueous space marker, carboxyfluorescein, when irradiated for 30‐120 s (average light intensity 50 W/m2). Liposomes not irradiated showed much lower rates of temperature dependent release. Such liposomes may be useful as biochemical tools or drug delivery systems in vitro and in vivo.