Platelet activating factor induces transient blood-brain barrier opening to facilitate edaravone penetration into the brain.

The blood-brain barrier (BBB) greatly limits the efficacy of many neuroprotective drugs' delivery to the brain, so improving drug penetration through the BBB has been an important focus of research. Here we report that platelet activating factor (PAF) transiently opened BBB and facilitated neuroprotectant edaravone penetration into the brain. Intravenous infusion with PAF induced a transient BBB opening in rats, reflected by increased Evans blue leakage and mild edema formation, which ceased within 6 h. Furthermore, rat regional cerebral blood flow (rCBF) declined acutely during PAF infusion, but recovered slowly. More importantly, this transient BBB opening significantly increased the penetration of edaravone into the brain, evidenced by increased edaravone concentrations in tissue interstitial fluid collected by microdialysis and analyzed by Ultra-performance liquid chromatograph combined with a hybrid quadrupole time-of-flight mass spectrometer (UPLC-MS/MS). Similarly, incubation of rat brain microvessel endothelial cells monolayer with 1 µM PAF for 1 h significantly increased monolayer permeability to (125)I-albumin, which recovered 1 h after PAF elimination. However, PAF incubation with rat brain microvessel endothelial cells for 1 h did not cause detectable cytotoxicity, and did not regulate intercellular adhesion molecule-1, matrix-metalloproteinase-9 and P-glycoprotein expression. In conclusion, PAF could induce transient and reversible BBB opening through abrupt rCBF decline, which significantly improved edaravone penetration into the brain. Platelet activating factor (PAF) transiently induces BBB dysfunction and increases BBB permeability, which may be due to vessel contraction and a temporary decline of regional cerebral blood flow (rCBF) triggered by PAF. More importantly, the PAF induced transient BBB opening facilitates neuroprotectant edaravone penetration into brain. The results of this study may provide a new approach to improve drug delivery into the brain.

The concordance of MRI and quantitative autoradiography estimates of the transvascular transfer rate constant of albumin in a rat brain tumor model.

The apparent forward transfer constant, K transa, for albumin was measured in 9L cerebral tumors in 15 rats. An MRI study using gadolinium-labeled bovine serum albumin was followed by terminal quantitative autoradiography (QAR) using radioiodinated serum albumin. Look-Locker MRI estimates of T(1) followed gadolinium-labeled bovine serum albumin blood and tissue concentration. QAR and MRI maps of K transa were coregistered, a region of interest (ROI) that included the tumor and its surround was selected, and the two estimates of K transa from the ROI on QAR and MRI maps were compared by either mean per animal ROI or on pixel-by-pixel data using a generalized estimating equation. An ROI analysis showed a moderate correlation between the two measures (r = 0.57, P = 0.026); pixel-by-pixel generalized estimating equation analysis concurred (r = 0.54, P < 0.0001). The estimates of QAR with MRI of last time points (e.g., 25 min) showed a moderate correlation (ROI r = 0.55, P < 0.035; generalized estimating equation r = 0.58, P < 0.0001). Differences between the QAR and MRI estimates of K transa did not differ from zero, but the MRI 25-min estimate was significantly lower than the QAR estimate. Thus, noninvasive MRI estimates of vascular permeability can serve as a surrogate for QAR measures.

Pinocytosis in Acanthamoeba castellanii. Kinetics and morphology.

The uptake of radioactively labeled albumin, inulin, leucine, and glucose by Acanthamoeba castellanii (Neff strain) was measured. The uptake is linear with time and appears to be continuous under the conditions of these experiments. Uptake is abolished at 0 degrees C. No evidence for saturation of the uptake mechanism was obtained with either albumin or leucine. Each of the four tracer molecules enters the ameba at a similar rate when the uptake is calculated as volume of fluid ingested per unit time. The data suggest that each of these molecules enters the cell by pinocytosis. The highest rate of uptake was obtained with cells in their usual culture medium containing proteose peptone, glucose, and salts but pinocytosis also continued at a reduced rate in a simple salt solution. The calculated volume of fluid taken in during pinocytosis in culture medium was about 2 microl/hr per 10(6) cells. The route of uptake was examined in the electron microscope using horseradish peroxidase (HRP) as a tracer. HRP activity was found exclusively within membrane profiles within the cytoplasm, confirming the pinocytotic mode of uptake. An estimate of the rate of surface membrane turnover due to pinocytosis was made using the biochemical and morphological data obtained. This estimate suggests that the plasma membrane turnover of one cell is on the order of several times an hour.

Structure-function relationships in the adipose cell. II. Pinocytosis and factors influencing its activity in the isolated adipose cell.

Pinocytic activity in the adipose cell has been examined by measuring the uptake of colloidal gold. Pinocytic activity occurs in the isolated adipose cell under all experimental conditions; a portion of the vesicular elements of the cell can be identified by electron microscopy as pinocytic in origin. The isolated adipose cell appears to take up serum albumin by pinocytosis. Pinocytic activity in the isolated adipose cell is enhanced by epinephrine, but not by insulin. The relationship between pinocytosis and the metabolic activity of the adipose cell has been studied by measuring simultaneously the uptake of radioactive colloidal gold, the incorporation of (14)C-counts from U-glucose-(14)C into CO(2), total lipid, triglyceride glycerol and triglyceride fatty acids, and the release of nonesterified fatty acids in the absence of hormones and in the presence of insulin or epinephrine. Correlations between hormone-produced alterations in lipid metabolism and in pinocytic activity suggest that intracellular nonesterified fatty acid levels are a factor in the regulation of both the cell's pinocytic activity and its metabolism and that pinocytosis in the adipose cell functions in the extracellular-intracellular transport of nonesterified fatty acids.

Studies on protein uptake by isolated tumor cells. 3. Apparent stimulations due to pH, hypertonicity, polycations, or dehydration and their relation to the enhanced penetration of infectious nucleic acids.

Fixation of (131)I-serum albumin by Ehrlich ascites tumor cells in suspensions and sarcoma S-180 monolayers was measured under experimental conditions. Anaerobic incubation and inhibitors of the oxidative metabolism critically restricted the range of glucose concentrations capable of supporting cell life; in glucose concentrations higher than 10(-2)M, Ehrlich cells suffered from their own acid production; in concentrations 10(-2)M, lower than they underwent damage by starvation. Both types of damage were accompanied by increased albumin fixation unrelated to pinocytosis. Different procedures recommended to enhance the uptake of infectious viral RNA by animal cells in culture were tested for their ability to increase albumin uptake. They enhanced the penetration of both albumin and vital dyes and decreased the viability of cell populations. Their effect, therefore, is related to cell damage. It was postulated that reversible damage to cells favors RNA infection by leading to abnormal uptake processes and by decreasing intracellular digestion. This abnormal uptake is different from pinocytosis and also from the massive fixation of albumin to dead cells. The latter phenomenon is due to adsorption by intracellular sites exposed by disruption of the cell membrane. Polycations are able to induce all three forms of fixation depending on the experimental conditions.

Uptake of protein by mammalian cells: an underdeveloped area. The penetration of foreign proteins into mammalian cells can be measured and their functions explored.

Although it is accepted on the basis of biological and morphological evidence that mammalian cells will take up macromolecules, little is known about the kinetics, the specificity, and the functions of this uptake. With labeled proteins used as models, it is found that the transport proceeds at very low rates, requires little energy, and is markedly enhanced by polybasic compounds. Molecular charge and size are important factors: cells clearly favor cationic macromolecules of large molecular weights. Neither factor, however, can fully account for the selectivity detected in the uptake of different proteins. Ingested albumin undergoes rapid and extensive degradation. This fact suggests that macromolecules have only a limited chance to express their biological activity in target cells, a finding that is relevant also to the role of foreign nucleic acids and the possibility of achieving genetic transformation in animal cells. There are concrete indications, however, that in spite of their short half-life, proteins can act as carriers, as precursors of active agents, and as regulators of metabolic functions in host cells. They may also be important in the control of growth and differentiation. These functions of exogenous proteins are still largely unexplored.

Absorption of inhaled antigen into the circulation of isolated lungs from normal and immunized rabbits.

The purpose of this study was to determine whether the absorption of inhaled antigen (Ag) across the pulmonary air-blood barrier of the isolated perfused lung can be modulated by immunologic mechanisms. Lungs from immunized or nonimmunized rabbits were removed, ventilated, and perfused with autochthonous blood. Radioiodinated Ag (human serum albumin or ovalbumin) was introduced as an aerosol into the isolated lung for 15 min and blood samples were taken over a 4-h period. The results showed that radioactivity fom inhaled Ag entered the perfusing blood as two fractions. One fraction was precipitable by 5% trichloroacetic acid or antiserum. The TCA-soluble fraction chromatographed differently from iodide and may have represented metabolites of the Ag. Immunization specifically reduced the amount of antigenically intact protein entering the blood. On the other hand, the metabolite reached higher concentrations in the blood of immunized lungs. We conclude that the alveolar capillary barrier of the normal rabbit lung could provide a significant route of entry for inhaled antigen into the systemic circulation and that immunization reduces absorption via this route and enhances pulmonary metabolism of the Ag.

Mechanism of acute depletion of plasma fibronectin following thermal injury in rats. Appearance of a gelatinlike ligand in plasma.

Plasma fibronectin was depleted within 15 min following sublethal burn, followed by partial recovery at 8 h and complete restoration by 24 h in anesthetized rats. Radiolabeled 75Se-plasma fibronectin, injected intravenously before burn, was rapidly sequestered in burn skin as well as the liver. Fibronectin levels at 2 h postburn as detected by immunoassay vs. 75Se-plasma fibronectin indicated that more fibronectin was in the plasma than detected by electroimmunoassay. Crossed immunoelectrophoretic analysis of fibronectin in early postburn plasma demonstrated a reduced electrophoretic mobility of the fibronectin antigen. Addition of heparin or fibrin, both of which have affinity for fibronectin, to normal plasma was unable to reproduce this altered fibronectin electrophoretic pattern. In contrast, addition of gelatin or native collagen to normal plasma reproduced the abnormal electrophoretic pattern of fibronectin seen in burn plasma. Extracts of burned skin, but not extracts of normal skin, when added to normal plasma, elicited a similar altered electrophoretic pattern for fibronectin. By gel filtration, fibronectin in burn plasma had an apparent molecular weight approximately 40% greater than that observed in normal plasma. These data suggest the release into the blood of a gelatinlike ligand from burned skin, which complexes with plasma fibronectin. Thus, fibronectin deficiency acutely postburn appears mediated by (a) its accumulation at the site of burn injury; (b) its removal from the circulation by the liver; and (c) its presence in the plasma in a form that is less detectable by immunoassay.

Induction of arthritis by purified cell-derived chemotactic factor: role of chemotaxis and vascular permeability.

The injection of monosidium urate-induced chemotactic factor into the joint cavities of rabbits induces an acute inflammatory response that resembles the one produced by monosodium urate crystals. The leukocyte accumulation induced by the factor was not accompanied by a measurable increase in vascular permeability as measured by appearance of 125I-albumin in the joint cavity. When histamine was injected into the joints, a marked increase in vascular permeability but no leukocytosis above control levels was observed. The above results suggest that the cell-derived factor is primarily responsible for the accumulation of cells seen in the acute inflammation induced by monosodium urate crystals.

Hypogammaglobulinemia associated with accelerated catabolism of IgG secondary to its inteaction with an IgG-reactive monoclonal IgM.

Hypogammaglobulinemia due to a new pathophysiological mechanism was studied in a patient with Sjögren's syndrome, a monoclonal IgM and a mixed (IgM-IgG) cryoglobulinemia. The IgM (IgMdk) component of the cryogel possessed light chains of lambda-type with highly restricted electrophoretic mobility analagous to those of a Waldenström's macroglobulin. IgMdk reacted specifically with native IgG, with IgG subclasses 1, 2, and 4, and with the Fc piece of IgG to form a cryogel. Serum concentrations of IgG 1, 2, and 4 were 10% of normal, whereas the IgG3 level was slightly increased and the IgM level was markedly increased. Viscosity and analytical ultracentrifugation studies with the purified mixed cryogel (IgM-LgG) indicated soluble complex formation over a temperature range (36-38 degrees C) attainable in vivo. Immunoglobulin turnover studies revealed a markedly elevated rate of IgM synthesis with a normal survival of IgM, IgA, and IgE. IgG3, which failed to form complexes with IgMdk at body temperature, had a normal synthetic rate and survival. In contrast, the other IgG subclasses showed reduced synthesis and shortened survival. These studies are the first indicating a short survival of some IgG subclasses with a normal survival of another. The hypogammaglobulinemia appears to be due in part to a new mechanism of accelerated protein catabolism: The rapid elimination of IgG due to its interaction with an IgG-reactive monoclonal IgM.

Identification and characterization of subpopulations of lymphocytes in human peripheral blood after fractionation on discontinuous gradients of albumin. The cellular defect in X-linked agammaglobulinemia.

Normal human peripheral blood lymphocytes were separated on discontinuous gradients of 17-35% bovine serum albumin (BSA) into nine fractions. Three subpopulations of lymphocytes were obtained. One occupies the top third of the gradient (fractions 1-3, 17-23% BSA) and is rich in cells characterized by a high spontaneous rate of DNA synthesis and by the ability to give rise to colony-forming units. The middle portion of the gradient (fractions 4 and 5, 23-27% BSA) is rich in thymus-derived (T) lymphocytes identified by their vigorous response to mitogens and by their ability to form rosettes with sheep erythrocytes (E). The third subpopulation at the bottom of the gradient (fractions 6-9, 27-35% BSA) is rich in bone marrow-derived (B) lymphocytes capable of staining with fluorescent antiimmunoglobulin antisera and of forming rosettes with EAC1423. The peripheral blood lymphocytes of five boys with proved X-linked agammaglobulinemia and two with probable X-linked agammaglobulinemia were found to be totally deficient in B lymphocytes (fractions 6-9) and lacked the subpopulation identified by immunofluorescent staining or rosette formation with EAC1423. One boy with proved X-linked agammaglobulinemia and two with probable X-linked agammaglobulinemia possessed a normal amount of circulating B lymphocytes.

Hepatic bilirubin uptake in the isolated perfused rat liver is not facilitated by albumin binding.

Bilirubin uptake by the liver is a rapid process of high specificity that has kinetic characteristics which suggest carrier-mediation. In the circulation, bilirubin is readily bound to albumin, from which it is extracted by the liver. Although several studies suggested that it is the small, unbound fraction of bilirubin which interacts with hepatocytes and is removed from the circulation, recent experiments have been interpreted as suggesting that binding to albumin facilitates ligand uptake. A liver cell surface receptor for albumin has been postulated. The present study was designed to examine directly whether albumin facilitates the hepatic uptake of bilirubin and whether uptake of bilirubin depends on binding to albumin. Rat liver was perfused with a protein-free fluorocarbon medium, and single-pass uptake of 1, 10, or 200 nmol of [3H]bilirubin was determined after injection as an equimolar complex with 125I-albumin, with 125I-ligandin, or free with only a [14C]sucrose reference. Uptake of 10 nmol of [3H]bilirubin was 67.5 +/- 3.7% of the dose when injected with 125I-albumin, 67.4 +/- 6.5% when injected with 125I-ligandin, and 74.9 +/- 2.4% when injected with [14C]sucrose (P greater than 0.1). At 200 nmol, uptake fell to 46.4 +/- 3.1% (125I-albumin) and 63.3 +/- 3.4% [( 14C]sucrose) of injected [3H]bilirubin (P less than 0.01), which suggests saturation of the uptake mechanism. When influx was quantitated by the model of Goresky, similar results were obtained. When [3H]bilirubin was injected simultaneously with equimolar 125I-albumin and a [14C]sucrose reference, there was no delay in 125I-albumin transit as compared with that of [14C]sucrose. This suggested that the off-rate of albumin from a putative hepatocyte receptor would have to be very rapid, which is unusual for high affinity receptor-ligand interaction. There was no evidence for facilitation of bilirubin uptake by binding to albumin or for interaction of albumin with a liver cell surface receptor. These results suggest that the hepatic bilirubin uptake mechanism is one of high affinity which can extract bilirubin from circulating carriers such as albumin, ligandin, or fluorocarbon.

Metabolism of L-thyroxine by phagocytosing human leukocytes.

Intact normal human leukocytes deiodinated L-thyroxine (T(4)) with the generation of inorganic iodide, chromatographically immobile origin material, and small quantities of L-triiodothyronine (T(3)). When phagocytosis was induced in the leukocytes through the addition of zymosan particles that had been opsonized by coating with plasma, T(4)-deiodination was greatly stimulated. In addition to the stimulation of T(4)-deiodination, the accumulation by the leukocytes of undegraded T(4) was increased. Anoxia, which has previously been shown not to interfere with phagocytosis, did not prevent the increased cellular accumulation of T(4) that phagocytosis induced, but virtually abolished T(4)-deiodination. On the other hand, calcium, which has previously been shown to be required for optimal phagocytosis, was required for the increase in both the cellular accumulation and deiodination of T(4) that phagocytosis induced. Phospholipase-C, which has previously been shown to induce a metabolic burst that mimics that induced by phagocytosis, did not increase the cellular accumulation or deiodination of T(4). On the other hand, colchicine, which has previously been shown to depress the metabolic burst that accompanies phagocytosis, did not prevent the increase in either the cellular accumulation or deiodination of T(4) that phagocytosis induced. Thus, increased accumulation of T(4) by the leukocytes during phagocytosis appears to be the primary factor responsible for the stimulation of deiodination that phagocytosis induces. The increased accumulation of T(4) did not appear to be owing to engulfment of suspending medium surrounding the particles or to binding of T(4) to the particles themselves. In addition to the enhanced cellular accumulation, other factors related to the metabolic burst that accompanies phagocytosis might also be involved in the stimulation of T(4)-deiodination. In leukocytes from two patients with chronic granulomatous disease, a disorder in which phagocytosis appears to occur normally but in which the metabolic burst and attendant increase in hydrogen peroxide generation do not occur, stimulation of T(4)-deiodination was either greatly diminished or totally lacking. In myeloperoxidase-deficient leukocytes, on the other hand, stimulation of T(4)-deiodination was at least as great as that in normal cells. Thus, we conclude that the primary factor responsible for the increased deiodination of T(4) that phagocytosis induces is the enhanced cellular uptake of hormone. The increased generation of hydrogen peroxide that accompanies phagocytosis may be necessary for the enhanced deiodination of the accumulated T(4), but the latter reaction does not require the mediation of myeloperoxidase.

Pulmonary microvascular dysfunction and pathological changes induced by blast injury in a rabbit model.

BACKGROUND: Vascular leakage has been proven to play a critical role in the incidence and development of explosive pulmonary barotrauma. Quantitatively investigated in the present study was the severity of vascular leakage in a gradient blast injury series, as well as ultrastructural evidence relating to pulmonary vascular leakage. METHODS: One hundred adult male New Zealand white rabbits were randomly divided into 5 groups according to distance from the detonator (10 cm, 15 cm, 20 cm, 30 cm, and sham control). Value of pulmonary vascular leakage was monitored by a radioactive 125I-albumin labeling method. Pathological changes caused by the blast wave were examined under light and electron microscopes. RESULTS: Transcapillary escape rate of 125I-albumin and residual radioactivity in both lungs increased significantly at the distances of 10 cm, 15 cm, and 20 cm, suggesting increased severity of vascular leakage in these groups. Ultrastructural observation showed swelling of pulmonary capillary endothelial cells and widened gap between endothelial cells in the 10-cm and 15-cm groups. CONCLUSION: Primary blast wave can result in pulmonary capillary blood leakage. Blast wave can cause swelling of pulmonary capillary endothelial cells and widened gap between endothelial cells, which may be responsible for pulmonary vascular leakage.

Interaction of phospholipid vesicles with rat hepatocytes in primary monolayer culture.

We studied the interaction of positively and negatively charged unilamellar and multilamellar phospholipid vesicles (liposomes) with rat-liver parenchymal cells in primary monolayer culture. Radioactive liposomal phosphatidylcholine was taken up more rapidly and to a larger extent from unilamellar than from multilamellar vesicles. No significant difference in uptake characteristics was observed between vesicles of different charge. The presence of serum greatly reduced uptake of liposomal phosphatidylcholine of both unilamellar and multilamellar vesicles. This serum effect was independent of surface charge of the vesicles. When cells were allowed to take up radioactive liposomal phospholipid and then incubated further in absence of vesicles, part of the radioactivity associated with the cells was released into the medium, most of it as water soluble degradation products. When cells were preincubated with vesicles containing horseradish peroxidase and then, after removal of the vesicles, further incubated, peroxidase activity could be demonstrated in the culture medium, part of it only after addition of Triton X-100. These observations were taken to indicate that part of the phospholipid taken up the cells represented vesicles binding to the cell surface rather than having been internalized. Vesicle-entrapped [125I]albumin was taken up by the cells and rapidly hydrolyzed as indicated by the appearance of radioactivity soluble in trichloroacetic acid within minutes after starting the incubation. No uptake of free albumin could be demonstrated. The kinetics of albumin uptake and release of trichloroacetic acid-soluble radioactivity from the cells suggest that, initially, liposomes are internalized predominantly by endocytosis, while during prolonged incubation fusion of the liposomal membrane with the plasma membrane gradually contributes more substantially to the overall uptake process. The significance of these findings is emphasized with special reference to the use of liposomes as intravenous carriers of enzymes or drugs.

Uptake of asialoglycophorin-liposomes by the perfused rat liver.

Asialoglycophorin-containing liposomes and their contents (125I-labeled bovine serum albumin) were taken up by a perfused rat liver with subsequent digestion of their protein components. The uptake of these liposomes required Ca2+ as well as desialylation. The process was inhibited partially by asialofetuin and completely by further addition of asialoglycophorin to the perfusate.

Rapid uptake by liver sinusoidal cells of serum albumin modified with retention of its compact conformation.

The clearance from the blood and the conformation of serum albumin modified by nitroguanidination and labeled with 125-I have been studied. Like formaldehyde-denatured albumin, but in contrast to native albumin, the nitroguanidinated derivative is rapidly cleared from the blood and taken up in lysosomes of liver sinusoidal cells. Although 94% of the free amino groups were blocked by nitroguanidination, we could not detect significant conformational changes using gel filtration, determination of reducible disulfide groups, and titration of tyrosine residues. It is concluded that extensive denaturation is no prerequisite for the uptake of albumin derivatives in liver sinusoidal cells. It is suggested that the nitroguanidinated protein, in contrast to native albumin, is bound on membrane receptors of sinusoidal cells. The nitroguanidino groups themselves might be bound on these receptors, but it seems equally possible that the blocking of positive charges of the albumin molecule or minor, local conformational changes of the protein are sufficient for the binding on the receptors.

Temperature-responsive size-exclusion chromatography using poly(N-isopropylacrylamide) grafted silica.

Silica-based packing materials induce non-specific interactions with proteins in aqueous media because of the nature of their surface, mainly silanol groups. Therefore, the silica surface has to be modified in order to be used as stationary phase for the High Performance Size-Exclusion Chromatography (HPSEC) of proteins. For this purpose, porous silica beads were coated with hydrophilic polymer gels (dextrans of different molecular weights) carrying a calculated amount of diethyl-aminoethyl groups (DEAE). Actually, as shown by HPSEC, these dextran modified supports minimize non-specific adsorption for proteins and pullulans in aqueous solution. Then, in order to change the pore size in response to temperature, temperature responsive polymer of poly(N-isopropylacrylamide) (PIPAAm) was introduced into the surface of dextran-DEAE on porous silica beads. The structure of these supports before and after modification was alternately studied by Scanning Electronic Microscopy (SEM) and Scanning Force Microscopy (SFM). An adsorption of radiolabelled albumin was performed to complete our study. Silica modifications by dextran-DEAE and PIPAAm improve the neutrality of the support and minimize the non-specific interactions between the solid support and proteins in solution. At low temperature, the support having PIPAAm exhibits a high resolution domain in HPSEC and finally permits a better resolution of proteins and pullulans. At higher temperature, hydrophobic properties of PIPAAm produce interactions with some proteins and trigger off a slight delay of their elution time.

Uptake and degradation of formaldehyde-treated 125I-labelled human serum albumin in rat liver cells in vivo and in vitro.

The uptake of formaldehyde-treated 125I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20-30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.

The permeability coefficient of albumin of the isolated rat mesentery. Modification by some mediators of inflammation, cyclic AMP and calcium.

In order to study the mechanisms whereby mediators of inflammation exert their exudative effects, we used isolated rat mesentery placed as a separation membrane between the two compartments of a diffusion cell. In this experimental arrangement, the permeability coefficient of albumin (PA) can be easily computed from the equilibration rate of 125I-labelled albumin added to one compartment. Histamine, bradykinin, serotonin and prostaglandins A1, A2, E1, E2, F1 alpha and F2 alpha all increased PA to some extent, the maximal values being approx. +60%. Dibutyryl-cyclic AMP, theophylline and isoproterenol also increased PA, thus suggesting involvement of cyclic AMP. Direct measurements of this nucleotide confirmed this hypothesis; furthermore, a linear relation between cyclic AMP levels and PA could be demonstrated. In contrast, cyclic GMP is probably not involved in the control of PA. Calcium-depleted tissues had a low PA (approx. 40% below controls), and did not respond to exogenous prostaglandin E1. These results suggest that transmesenteric passage of albumin is at least partly controlled by cyclic AMP and intracellular Ca2+ levels.