Extracellular Albumin Research Articles

Protein-aggregating ability of different protoporphyrin-IX nanostructures is dependent on their oxidation and protein-binding capacity

Porphyrias are rare blood disorders caused by genetic defects in the heme biosynthetic pathway and are associated with the accumulation of high levels of porphyrins that become cytotoxic. Porphyrins, due to their amphipathic nature, spontaneously associate into different nanostructures, but very little is known about the cytotoxic effects of these porphyrin nanostructures. Previously, we demonstrated the unique ability of fluorescent biological porphyrins, including protoporphyrin-IX (PP-IX), to cause organelle-selective protein aggregation, which we posited to be a major mechanism by which fluorescent porphyrins exerts their cytotoxic effect. Herein, we tested the hypothesis that PP-IX-mediated protein aggregation is modulated by different PP-IX nanostructures via a mechanism that depends on their oxidizing potential and protein-binding ability. UV–visible spectrophotometry showed pH-mediated reversible transformations of PP-IX nanostructures. Biochemical analysis showed that PP-IX nanostructure size modulated PP-IX-induced protein oxidation and protein aggregation. Furthermore, albumin, the most abundant serum protein, preferentially binds PP-IX dimers and enhances their oxidizing ability. PP-IX binding quenched albumin intrinsic fluorescence and oxidized His-91 residue to Asn/Asp, likely via a previously described photo-oxidation mechanism for other proteins. Extracellular albumin protected from intracellular porphyrinogenic stress and protein aggregation by acting as a PP-IX sponge. This work highlights the importance of PP-IX nanostructures in the context of porphyrias and offers insights into potential novel therapeutic approaches.

Yap/Taz promote the scavenging of extracellular nutrients through macropinocytosis.

The uptake of macromolecules and cellular debris through macropinocytosis has emerged as an important nutrient acquisition strategy of cancer cells. Genetic alterations commonly found in human cancers (e.g. mutations in KRAS or loss of PTEN) have been shown to increase macropinocytosis. To identify additional effectors that enable cell growth dependent on the uptake of extracellular proteins, pancreatic ductal adenocarcinoma (PDA) cells were selected for growth in medium where extracellular albumin was the obligate source of the essential amino acid leucine. Analysis of global changes in chromatin availability and gene expression revealed that PDA cells selected under these conditions exhibited elevated activity of the transcriptional activators Yap/Taz. Knockout of Yap/Taz prevented growth of PDA cells in leucine-deficient medium, but not in complete medium. Furthermore, constitutively active forms of Yap or Taz were sufficient to stimulate macropinocytosis of extracellular protein. In addition to promoting the uptake of plasma proteins, Yap/Taz also promoted the scavenging of apoptotic cell bodies and necrotic debris by PDA cells. The Yap/Taz transcriptional target Axl was found to be essential for cell growth dependent on the uptake of dead cells and cell debris. Together, these studies suggest that the Hippo pathway effectors Yap and Taz are important transcriptional regulators of endocytic nutrient uptake.

Extracellular Albumin Covalently Sequesters Selenocompounds and Determines Cytotoxicity.

Selenocompounds (SeCs) are well-known nutrients and promising candidates for cancer therapy; however, treatment efficacy is very heterogeneous and the mechanism of action is not fully understood. Several SeCs have been reported to have albumin-binding ability, which is an important factor in determining the treatment efficacy of drugs. In the present investigation, we hypothesized that extracellular albumin might orchestrate SeCs efficacy. Four SeCs representing distinct categories were selected to investigate their cytotoxicity, cellular uptake, and species transformation. Concomitant treatment of albumin greatly decreased cytotoxicity and cellular uptake of SeCs. Using both X-ray absorption spectroscopy and hyphenated mass spectrometry, we confirmed the formation of macromolecular conjugates between SeCs and albumin. Although the conjugate was still internalized, possibly via albumin scavenger receptors expressed on the cell surface, the uptake was strongly inhibited by excess albumin. In summary, the present investigation established the importance of extracellular albumin binding in determining SeCs cytotoxicity. Due to the fact that albumin content is higher in humans and animals than in cell cultures, and varies among many patient categories, our results are believed to have high translational impact and clinical implications.

Extracellular Albumin and Endosomal Ions Prime Enterovirus Particles for Uncoating That Can Be Prevented by Fatty Acid Saturation.

There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-Å resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release.IMPORTANCE There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses.

KRAS-enhanced macropinocytosis and reduced FcRn-mediated recycling sensitize pancreatic cancer to albumin-conjugated drugs

Pancreatic ductal adenocarcinoma (PDAC) is a dominantly (~95%) KRAS-mutant cancer that has extremely poor prognosis, in part this is due to its strong intrinsic resistance towards almost all therapeutic agents. PDAC relies heavily on KRAS-transformed metabolism, including enhanced macropinocytosis and catabolism of extracellular albumin, to maintain its proliferation and progression. However, it has yet to be validated that whether such transformed metabolism could be exploited for the drug delivery to open therapeutic windows of cytotoxic agents in KRAS-mutant PDAC. In this study, we attempt to answer this question by focusing on the impact of two critical regulators of albumin catabolism, KRAS and the neonatal Fc receptor (FcRn), on the sensitivity of PDAC to doxorubicin (DOX, a model cytotoxic agent) and albumin-conjugated doxorubicin (DOX-ALB). Using cell lines and cell-derived xenografts with different KRAS genotypes and FcRn levels, we demonstrated that KRAS-enhanced macropinocytosis and reduced FcRn expression sensitize PDAC to DOX-ALB but not free DOX. In both in vitro and in vivo comparsion, the DOX-ALB demonstrated ~10 times enlarged therapeutic window compared with free DOX, in PDAC with KRAS mutation and reduced FcRn level, two events appear to occur simultaneously in the investigated PDAC. In summary, we conclude that albumin conjugation is an exploitable drug delivery strategy that significantly opens the therapeutic windows of otherwise undevelopable anti-cancer agents for KRAS-mutant PDAC therapy, and creates a new landscape for clinical evaluation and future translation of such compounds.

Possible Mechanisms by Which Enzymatic Degradation of Human Serum Albumin Can Lead to Bioactive Peptides and Biomarkers.

Partial enzymatic degradation of human serum albumin in vivo can lead to the generation of peptides with novel functions or to peptides that might serve as biomarkers for disease. In pathological conditions, biomarkers are possibly produced from the protein in the lysosomes and set free by cell death, or cell death could release acid endoproteases which produce biomarkers by degrading extracellular albumin. Alternatively, lysosomes or secretory granules can be stimulated to release enzymes which produce bioactive peptides from albumin. In physiological conditions, it is proposed that bioactive peptides can be made by enzymatic attack on the protein bound to the endosomal neonatal Fc receptor. The peptides formed could leave the cell, together with native albumin, by exocytosis. Thus, the receptor could have a new function in addition to saving albumin from degradation in the lysosomes. Large amounts of albumin are degraded every day, and this fact can compensate for the short in vivo half-lives of the bioactive peptides. One or more of the procedures outlined above could also apply to other plasma proteins or to structural proteins.

Transverse water relaxation in whole blood and erythrocytes at 3T, 7T, 9.4T, 11.7T and 16.4T; determination of intracellular hemoglobin and extracellular albumin relaxivities

Blood is a physiological substance with multiple water compartments, which contain water-binding proteins such as hemoglobin in erythrocytes and albumin in plasma. Knowing the water transverse (R2) relaxation rates from these different blood compartments is a prerequisite for quantifying the blood oxygenation level-dependent (BOLD) effect. Here, we report the Carr-Purcell-Meiboom-Gill (CPMG) based transverse (R2CPMG) relaxation rates of water in bovine blood samples circulated in a perfusion system at physiological temperature in order to mimic blood perfusion in humans. R2CPMG values of blood plasma, lysed packed erythrocytes, lysed plasma/erythrocyte mixtures, and whole blood at 3 T, 7 T, 9.4 T, 11.7 T and 16.4 T were measured as a function of hematocrit or hemoglobin concentration, oxygenation, and CPMG inter-echo spacing (τcp). R2CPMG in lysed cells showed a small τcp dependence, attributed to the water exchange rate between free and hemoglobin-bound water to be much faster than τcp. This was contrary to the tangential dependence in whole blood, where a much slower exchange between cells and blood plasma applies. Whole blood data were fitted as a function of τcp using a general tangential correlation time model applicable for exchange as well as diffusion contributions to R2CPMG, and the intercept R20blood at infinitely short τcp was determined. The R20blood values at different hematocrit and the R2CPMG values of lysed erythrocyte/plasma mixtures at different hemoglobin concentration were used to determine the relaxivity of hemoglobin inside the erythrocyte (r2Hb) and albumin (r2Alb) in plasma. The r2Hb values obtained from lysed erythrocytes and whole blood were comparable at full oxygenation. However, while r2Hb determined from lysed cells showed a linear dependence on oxygenation, this dependence became quadratic in whole blood. This possibly suggests an additional relaxation effect inside intact cells, perhaps due to hemoglobin proximity to the erythrocyte membrane. However, we cannot exclude that this is a consequence of the simple tangential model used to remove relaxation contributions from exchange and diffusion. The extensive data set presented should be useful for future theory development for the transverse relaxation of blood.

Specialty training applications for entry in 2016: competition ratios and the application process

Abstract In diseases of many parenchymatous organs, heterogenous detoriation of individual functional units determines the clinical prognosis. However, the molecular characterization of these subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Sclerotic and proteinuric glomerular kidney disease is a frequent and heterogeneous disease which affects a fraction of nephrons, glomeruli and draining tubules, to variable extents, and for which no treatment exists. Here, we developed and applied an antibody-independent methodology to investigate heterogeneity of individual nephron segment proteomes from mice with proteinuric kidney disease. This “one-segment-one-proteome-approach” defines mechanistic connections between upstream (glomerular) and downstream (tubular) nephron segment populations. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins. In an attempt to target this protein co-expression module, genetic ablation of LAMP1-correlated lysosomal proteases in mice could ameliorate glomerular damage. Furthermore, individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases demonstrated increased abundance of lysosomal proteins, in combination with a decreased abundance of the mutated gene products. Therefore, increased glomerular lysosomal load is a conserved key mechanism in proteinuric kidney diseases, and the technology applied here can be implemented to address heterogeneous pathophysiology in a variety of diseases at a sub-biopsy scale

Abstract PR08: Pulling out all the stops: Exploiting macropinocytosis inhibition for the treatment of pancreatic cancer

Abstract Oncogenic Ras stimulates macropinocytosis, an endocytic mechanism of fluid-phase uptake that produces large intracellular vesicles known as macropinosomes. Recently, we have linked the macropinocytic uptake of extracellular albumin and its subsequent degradation to amino acid supply and proliferation in Ras-transformed cells. The ability of albumin to serve as a nutrient source in oncogenic Ras-expressing cells is blocked by inhibiting its internalization via treatment with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). We determined that EIPA treatment diminished the growth of pancreatic tumor xenografts and that this effect was selective for tumors with a high macropinocytic index. Currently, we are exploring the feasibility of employing macropinocytosis inhibition as an anticancer therapeutic modality utilizing an autochthonous mouse model of pancreatic cancer. In these autochthonous tumors, macropinocytosis is a prominent feature of pancreatic cells found in mid- to late-stage PanIN lesions, as well as in fibroblasts and immune cells residing within the tumor stroma. We have found that EIPA treatment results in a rapid and robust reduction in proliferative capacity both in tumor cells and the surrounding stromal cells. Intriguingly, our preliminary data indicates that EIPA treatment reduces the number of activated fibroblasts associated with PanIN lesions, decreases collagen deposition and results in an increase in blood vessel diameter. Studies have demonstrated that targeting components of the extracellular matrix within the tumor stroma can cause expansion of the vasculature, which can be harnessed to improve drug delivery and permeability to the tumor. Altogether, our findings suggest that macropinocytosis inhibition could be exploited not only to target the tumor cells, but also to target the tumor stroma and enhance the delivery of chemotherapeutics. This abstract is also presented as Poster B37. Citation Format: Cosimo Commisso, Craig Ramirez, Rengin Soydaner-Azeloglu, David L. Bajor, Robert H. Vonderheide, Dafna Bar-Sagi. Pulling out all the stops: Exploiting macropinocytosis inhibition for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR08.

Determining the macropinocytic index of cells through a quantitative image-based assay

Macropinocytosis serves as an internalization pathway for extracellular fluid and its contents. Macropinocytosis is upregulated in oncogene-expressing cells and, recently, we have revealed a functional role for macropinocytosis in fueling cancer cell growth through the internalization of extracellular albumin, which is degraded into a usable source of intracellular amino acids. Assessing macropinocytosis has been challenging in the past because of the lack of reliable assays capable of quantitatively measuring this uptake mechanism. Here we describe a protocol for visualizing and quantifying the extent of macropinocytosis in cells both in culture and growing in vivo as tumor xenografts. By using this approach, the 'macropinocytic index' of a particular cell line or subcutaneous tumor can be ascertained within 1-2 d. The protocol can be carried out with multiple samples in parallel and can be easily adapted for a variety of cell types and xenograft or allograft mouse models.

Abstract PR12: Bringing the outside in: Macropinocytosis and cancer therapeutics

Abstract Oncogenic Ras mutations are prevalent in a variety of tumor types, including adenocarcinomas of the pancreas, colon, and lung. One of the most overt phenotypes associated with the expression of oncogenic Ras mutants is the stimulation of macropinocytosis, an endocytic process that involves extensive membrane remodeling and the internalization of extracellular fluid via large membrane-bound vesicles called macropinosomes. The functional consequences of this stimulation in oncogenic Ras-expressing cancer cells were unknown prior to our recent work where we linked macropinocytic uptake to nutrient delivery and amino acid supply in tumor cells. We found that Ras-transformed cells utilize macropinocytosis to internalize extracellular albumin, which is then lysosomally degraded releasing the constituent amino acids intracellularly. These protein-derived amino acids have the capacity to enter central carbon metabolism and fuel tumor cell proliferation even in a nutrient-depleted environment. Of particular relevance is the finding that the pharmacological inhibition of macropinocytosis compromises the growth of Ras-driven pancreatic xenograft tumors. This discovery raises the question of whether the inhibition of macropinocytosis can be utilized as a therapeutic intervention in a subset of cancers. Moreover, because macropinocytosis is an established mechanism of drug delivery for nanoparticles, our work highlights the possibility of exploiting this feature of Ras-induced tumors for the effective delivery of nanoscale therapeutics. Our recent studies focused on identifying novel modulators of macropinocytosis and examining macropinocytosis as a delivery mechanism for nab-paclitaxel, an albumin-based nanoparticle therapy, will be discussed. Citation Format: Cosimo Commisso, Venugopal Chenna, Elda Grabocka, Craig Ramirez, Maitra Anirban, Dafna Bar-Sagi. Bringing the outside in: Macropinocytosis and cancer therapeutics. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR12.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 5147: Glutamine addiction: Recognizing and harnessing the pathometabolic potential of Ras-mediated macropinocytosis

Abstract Oncogenic mutations in Ras-encoding genes are found in approximately 30% of all human tumors and are most prevalent in carcinomas of the pancreas, colon, lung and bladder. Oncogenic forms of Ras are trapped in a constitutively active state and, as a consequence, promote the persistent stimulation of effector pathways that control cell growth, survival and proliferation. A prominent phenotypic feature associated with oncogenic Ras expression is the stimulation of macropinocytosis, an endocytic process that mediates the uptake of extracellular fluid via large, heterogeneous vesicles known as macropinosomes. While in amoeboid organisms this process has been linked to nutrient internalization, the functional significance of oncogenic Ras-mediated macropinocytosis is unknown. We have found that cancer cells harboring oncogenic Ras utilize macropinocytosis to internalize extracellular albumin, a major constituent of extracellular fluids and a potentially rich source of amino acids. Furthermore, we have established that tumor cells harboring oncogenic Ras mutations display robust macropinocytosis in vivo. Utilizing cell biological approaches and amino acid quantification, we determined that extracellular albumin that is internalized via oncogenic Ras-stimulated macropinosomes is targeted for degradation leading to the intracellular production of albumin-derived amino acids. Cancer cells display a heightened dependence on glutamine due to their need to sustain rapid proliferation, bioenergetics and macromolecular biosynthesis. We have observed that in cells expressing oncogenic Ras, the adverse effects of glutamine deprivation on cell growth can be rescued by macropinocytosis-mediated uptake of extracellular albumin. Moreover, the capacity of macropinocytic inhibition to diminish this growth advantage is dependent on both glutamine and glutamine-derived metabolites. These results indicate that macropinocytosis may serve as a glutamine supply mechanism that is particularly critical under conditions where free glutamine is limiting. Together, these data implicate oncogenic Ras-mediated macropinocytosis in the regulation of cancer cell metabolism and point to the possibility of exploiting this process in the design of anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5147. doi:1538-7445.AM2012-5147

Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage and release upon oxidation of the extramitochondrial fraction of the flavonoid

Quercetin uptake in Jurkat cells is extremely rapid and associated with a remarkable accumulation of the flavonoid, dependent on its binding to intracellular components. Cell-associated quercetin is biologically active, quantitatively consumed to promote survival in the presence of reactive species, such as peroxynitrite (ONOO −), or reduction of extracellular oxidants via activation of plasma membrane oxidoreductases. In alternative, quercetin is very slowly released upon post-incubation in drug-free medium, an event significantly accelerated by extracellular albumin. Quercetin uptake is also observed in isolated mitochondria, resulting in an enormous accumulation of the flavonoid, consumed under conditions associated with prevention of lipid peroxidation induced by ONOO −. Interestingly, remarkable quercetin accumulation is also detected in the mitochondria isolated from quercetin-pre-loaded cells, and exposure to either ONOO − or extracellular oxidants caused the parallel loss of both the mitochondrial and cytosolic fractions of the flavonoid. In conclusion, Jurkat cells accumulate large amounts of quercetin and even larger amounts of the flavonoid further accumulate in their mitochondria. Intramitochondrial quercetin appears to be functional for prevention of mitochondrial damage as well as for redistribution to the cytosol, when the fraction of the flavonoid therein retained is progressively consumed either by cell-permeant oxidants or by activation of plasma membrane oxidoreductases.

Presence of plasma proteins in spinal nerve roots. An immunohistochemical study in the rat

The presence of plasma proteins in the spinal nerve roots of normal rats was investigated using an avidin-biotin peroxidase technique on formaldehyde-fixed, paraffin-embedded material. Sections from the roots, exposed to a rabbit-anti rat albumin antiserum showed widespread, intense immunoreactivity which filled the spaces between the nerve fibers. The reaction product usually ended at the junction between the roots and the spinal cord. The sheath enclosing the roots showed the same strong immunoreaction. There was also a marked reaction in the dorsal root ganglia and peripheral nerve. Spinal cord sections, however, showed no extracellular reactivity, but many motor neurons of the ventral horn were distinctly positive, presumably the result of a normally occurring retrograde axonal transport from the periphery. Parallel sections from the roots exposed to rabbit anti rat IgG antiserum, rabbit anti rat IgM antiserum, rabbit anti human fibrinogen antiserum and rabbit anti human fibronectin antiserum revealed no positive immunoreaction. Thus, rat spinal nerve roots normally contain material with albumin antigenic properties. This would indicate that albumin is present in the extracellular fluid of the roots in the same way as in the endoneurium of peripheral nerves. The fluid microenvironment of the roots, therefore, appears to be different from that in the CNS which lacks extracellular albumin due to the impermeability of the blood-brain barrier.

A simple non‐destructive method for the fixation and immunostaining of cultured cells encapsulated in alginate

In the present paper we report a simple non-destructive method for the analysis of cells and their proteins encapsulated in alginate. Investigations with a co-culture of the human hepatoma cell line HepG2 and primary human fibroblasts are reported. We studied the expression of three proteins, namely intracellular alpha-foetoprotein and extracellular albumin, expressed by HepG2, and membrane-bound CD90 (Thy-1), expressed by the fibroblasts. Fluorimetric and colorimetric staining methods were evaluated and compared. We optimized the method by investigating a range of concentrations of the washing buffers. The technique protected the capsule and cell structures and enabled the investigated proteins to be clearly and reliably visualized within the encapsulated co-culture in their respective intracellular, membrane-bound and extracellular domains. These results demonstrate that this simple method is suitable for the non-destructive analysis of protein expression by cells encapsulated in alginate.

The role of paf in embryo physiology

Embryo-derived paf (1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is produced by de novo synthesis. This synthesis commences soon after fertilization and persists throughout the preimplantation phase. Paf is produced and released by the embryos of all mammalian species studied to date. Its release from the embryo involves binding to extracellular albumin in a manner that protects paf from enzymatic degradation. Released paf causes a range of alterations in maternal physiology, including platelet activation, changes in oviductal, endometrial and maternal immune function. Paf also acts in an autocrine fashion as a trophic/survival factor for the early embryo. In vitro, supplementation of culture media with paf improves embryo development. Embryo-derived paf's autocrine actions are transduced by 1-o-phosphatidylinositol-3-kinase, which induces characteristic calcium transients within the early embryo. The calcium transients require both the influx of external calcium and release of inositol trisphosphate-dependent internal calcium stores. Buffering these transients compromised embryo development in a manner that was reversed by exogenous paf. Assisted reproductive technologies compromise the production of paf by some embryos and retard the expression of the paf receptor. This deprivation of paf's action is one of the factors limiting the survivability of embryos produced by assisted reproductive technologies. Paf is one of several autocrine and paracrine trophic/survival factors that act on the early embryo. These factors probably act cooperatively and may, to some degree, be mutually redundant. As the earliest-released and the best-described embryotrophin, paf provides an important exemplar for understanding the role of ligand-mediated trophic support of the early embryo.

Interstitial exclusion of albumin in rabbit lung during development of pulmonary oedema

The modifications of the macromolecular sieving properties of the pulmonary extracellular tissue matrix were studied in adult anaesthetized rabbits (n= 10) exposed to increased tissue hydration. Exclusion of albumin from the perivascular pulmonary interstitial space was determined by using the continuous infusion method coupled with direct sampling of interstitial fluid performed through the wick technique. The rabbits underwent an intravenous infusion of saline amounting to 10 (n= 5) or 20 % (n= 5) body weight. Extracellular albumin distribution volume was derived from the steady state tissue concentration of radioactive rabbit serum albumin (125I-RSA). Pulmonary extracellular and intravascular fluid volumes (Vx and Vv, respectively) were measured as distribution volumes of 51Cr-EDTA and 131I-RSA, respectively, and interstitial fluid tracer concentrations were determined in interstitial fluid collected through implanted wicks. At the highest degree of hydration, interstitial fluid volume (Vi=Vx−Vv) and extravascular albumin distribution volume (Va,w) significantly increased by 38.5 and 240.2 %, respectively, compared to control. Albumin-excluded volume (Ve,a=Vi−Va,w) was 398.9 ± 17 μl (g dry tissue weight)−1; the albumin-excluded volume fraction (Fe,a=Ve,a/Vi) was 0.23 ± 0.01, 33.2 % of the control value. Data indicate that, at variance with what is observed in tissues like skin and muscle, pulmonary Fe,a is highly sensitive to tissue fluid content.

Interstitial exclusion of albumin in rabbit lung measured with the continuous infusion method in combination with the wick technique.

To determine albumin exclusion from the pulmonary perivascular interstitium by using the continuous infusion methods coupled with direct samples of pulmonary interstitial fluid via the wick technique. To evaluate extracellular albumin distribution volume, radioactive ((125)I) rabbit serum albumin (RSA) was delivered intravenously until reaching a steady-state tissue concentration. In anesthetized rabbits with ligated kidney pedicles, extracellular (V(x)) and intravascular (V(v)) fluid volumes were measured as the distribution volumes of (51)Cr-ethylenediaminetetraacetic a id and (131)I-RSA, respectively. Post mortem, lung samples and interstitial fluid collected via implanted wicks were assayed for isotope activity. The extravascular wick fluid equivalent albumin distribution volume (V(a,w)) was calculated assuming equal concentrations of (125)I-RSA in interstitial and wick fluid. The mean (+/-SE) V(x), V(v), and interstitial fluid volumes (V(i) = V(x) - V(v)) were 1896 +/- 61 (n = 8), 556.8 +/- 19, and 1329 +/- 73 microL/g of blood-free dry lung tissue weight, respectively. The mean albumin-excluded volume (V(e,a) = V(i) - V(a,w) and the excluded fraction (V(e,a)/V(i)) were 875.8 microL/g +/- 58 and 0.66 +/- 0.05, respectively. At variance with previous indirect reports, the present data indicate that normal pulmonary parenchyma displays a tight fibrous structure that is highly restrictive with respect to plasma protein distribution.

Interstitial exclusion of albumin in rabbit lung during development of pulmonary oedema

The modifications of the macromolecular sieving properties of the pulmonary extracellular tissue matrix were studied in adult anaesthetized rabbits (n = 10) exposed to increased tissue hydration. Exclusion of albumin from the perivascular pulmonary interstitial space was determined by using the continuous infusion method coupled with direct sampling of interstitial fluid performed through the wick technique. The rabbits underwent an intravenous infusion of saline amounting to 10 (n = 5) or 20 % (n = 5) body weight. Extracellular albumin distribution volume was derived from the steady state tissue concentration of radioactive rabbit serum albumin (125I-RSA). Pulmonary extracellular and intravascular fluid volumes (Vx and Vv, respectively) were measured as distribution volumes of 51Cr-EDTA and 131I-RSA, respectively, and interstitial fluid tracer concentrations were determined in interstitial fluid collected through implanted wicks. At the highest degree of hydration, interstitial fluid volume (Vi = Vx - Vv) and extravascular albumin distribution volume (Va,w) significantly increased by 38.5 and 240.2 %, respectively, compared to control. Albumin-excluded volume (Ve,a = Vi - Va,w) was 398.9 +/- 17 microl (g dry tissue weight)-1; the albumin-excluded volume fraction (Fe,a = Ve,a/Vi) was 0.23 +/- 0.01, 33.2 % of the control value. Data indicate that, at variance with what is observed in tissues like skin and muscle, pulmonary Fe,a is highly sensitive to tissue fluid content.