Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver.

Open fenestrations are a conspicuous feature of sinusoidal endothelial cells and allow free movement of plasma into the space of Disse. In hepatic fibrosis, the number of fenestrations decreases as interstitial collagen increases in the liver, a change that correlates with deposition of extracellular matrix in the space of Disse. In this study, the possibility of a causal relationship between altered fenestral morphology and perisinusoidal matrix has been examined by culturing rat sinusoidal endothelial cells on individual matrix proteins or on a native matrix consisting of human amniotic membrane with interstitial collagen (types I and III) on one side and basement membrane proteins (collagen types IV and V and laminin) on the other. Under culture conditions, individual components of the extracellular matrix failed to maintain fenestrations. A basement-membranelike gel matrix derived from the Engelbreth-Holm-Swarm tumor war similarly ineffective. Fenestral density and porosity (percentage of cell surface occupied by fenestrations) were significantly enhanced, however, when endothelial cells were cultured on the basement-membrane side of human amnion. These data suggest that support of endothelial fenestrations requires a complex matrix. In particular, physiologically derived basement membrane maintains fenestrations, whereas interstitial collagen matrix does not. The loss of fenestrations associated with hepatic fibrosis may be related in part to an accumulation of interstitial collagens in the space of Disse.

Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture.

The development of techniques for isolating hepatic lipocytes (Ito, stellate or fat-storing cells) from rodents has been instrumental in defining their role in hepatic vitamin A storage and fibrogenesis. In this study, we developed a method for the purification of lipocytes and Kupffer cell from wedge sections of normal human liver and examined their properties in primary culture. Sections of donor liver (400 to 600 gm) harvested but not used for transplantation were perfused in situ with University of Wisconsin solution and used for lipocyte isolation within 48 hr. Cells were isolated by catheter perfusion of the wedge through several large vessels with L-15 salts, Pronase and collagenase, followed by Larex density gradient centrifugation. Lipocytes were plated on either uncoated plastic or a basement membrane-like gel. Lipocyte and Kupffer cell yields were 2.3 +/- 0.6 x 10(5) and 8.6 +/- 1.4 x 10(5) cells, respectively, per gram of liver (n = 5). Lipocyte purity was 91% as assessed by vitamin A autofluorescence, and Kupffer cell purity was 83% as determined by uptake of fluorescinated staphylococci. Lipocytes cultured on the plastic spread within 48 to 72 hr, displaying slightly more heterogeneous retinoid droplet size than comparable rat cells; on a basement-membrane gel, the cells remained aggregated and spherical with occasional spindlelike extensions. Lipocytes on plastic expressed procollagens I and III, collagen IV and laminin by immunocytochemistry, and types I, III and IV procollagen messenger RNAs by RNAse protection. Northern blot and polymerase chain reaction, respectively. Transmission electron microscopy of lipocytes at 7 days demonstrated a prominent rough endoplasmic reticulum and contractile filaments. Scanning electron microscopy revealed a smooth cell surface with perinuclear droplets beneath the cell membrane. With continued primary culture on plastic (more than 7 days), cells appeared "activated" (i.e., increased spreading and diminished retinoid droplets) and began proliferating as assessed by nuclear autoradiography and [3H]thymidine incorporation. Kupffer cells observed by scanning electron microscopy in early primary culture displayed prominent membrane ruffling and lamellipodia. In summary, we have established a reproducible method for the isolation and primary culture of human lipocytes and Kupffer cells.

Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo.

Whether parenchymal or nonparenchymal liver cells play a predominant role in the pathophysiology of hepatic fibrosis has not been firmly established in vivo. We have addressed this question by quantitating the relative abundance of specific mRNAs for collagen types I, III, and IV, and laminin in purified populations of hepatocytes, sinusoidal endothelial cells, and lipocytes from normal and fibrotic rat liver. In normal liver, type I collagen gene expression was minimal in all cell types; mRNA for types III and IV collagen were apparent in endothelial cells and lipocytes, but not in hepatocytes. Laminin mRNA was present in all cell types. Induction of fibrogenesis by either bile duct ligation or carbon tetrachloride administration was associated with a substantial increase in mRNA for types I and III collagen in nonparenchymal cells. Lipocytes from fibrotic animals exhibited a greater than 30-fold increase in type I collagen mRNA relative to normal lipocytes, and greater than 40-fold relative to hepatocytes. Type III collagen mRNA reached 5 times that in normal lipocytes and greater than 120 times that in hepatocytes. Endothelial cells exhibited an isolated increase in type I collagen mRNA, reaching five times that in normal liver. Type IV collagen and laminin gene expression were not significantly increased in nonparenchymal cells during fibrogenesis; in fact, mRNA for type IV collagen and laminin decreased by up to 50% in endothelial cells. Despite the pronounced changes that occurred in matrix gene expression in nonparenchymal cells during fibrogenesis, no change was noted in hepatocytes. We conclude that nonparenchymal liver cells, particularly lipocytes, are important effectors of hepatic fibrosis in vivo.

Corticosteroid-induced pancreatitis: a case report demonstrating recurrence with rechallenge.

The existence of corticosteroid-induced pancreatitis remains controversial, despite the fact that more than 40 cases have been reported since its first description in 1955. No previous case reports have shown recurrence of pancreatitis after rechallenge with corticosteroids. This report describes a patient with stage IIIB Hodgkin's disease who received dexamethasone on three occasions for symptoms of spinal cord compression. On each occasion, the patient developed clinically evident pancreatitis shortly after beginning corticosteroid therapy. We believe that the close temporal relationship of the recurrences following rechallenge clearly implicates corticosteroids as an etiologic factor in this patient's pancreatitis.

Distortion of cyclic AMP responses to catecholamine due to destruction of the hormone.

The acute actions of low and moderate concentrations of catecholamines on cyclic AMP metabolism in SV40-transformed human lung fibroblasts (VA13) were seriously distorted by non-enzymatic destruction of the agonist. Catecholamine destruction, as measured directly with an isotopic method, was slowed by a variety of anti-oxidants and chelating agents. A combination of two anti-oxidants, ascorbate and thiourea, was very effective in protecting isoproterenol in the cell culture system. That is, there was a 10-fold increase in the sensitivity of VA13 to isoproterenol and the duration of action of the catecholamine was greatly prolonged. However, the anti-oxidants did not alter the responses of the cells to prostaglandins. We conclude that any quantitative studies of cyclic AMP responses to catecholamines must address the question of agonist destruction if meaningful results are to be expected. The use of anti-oxidants, especially the combination of ascorbate and thiourea, would appear to be advisable, particularly in situations where the catecholamine concentrations are less than supramaximal.

Glycoprotein-enriched vesicles from sheep erythrocyte ghosts obtained by spontaneous vesiculation.

Sheep erythrocyte membranes have been shown in this laboratory to undergo spontaneous vesiculation when incubated at 4 degrees, fractionating into two bands in dextran gradients (R. McGuire and R. Barber, submitted for publication). While vesicles were observed to be formed in several solvent systems, incubation in the presence of complexors to remove divalent cations was found to be the most efficient method for both vesicle formation and their detachment from the residual membrane. We report here on the characterization of these vesicles formed by spontaneous vesiculation. In the presence of a hypotnoic buffer containing 1 mM EDTA, vesicle production proceeds linearly up to 50 hours and declines, reaching its maximum at 72 hours with up to 20% of the total membrane protein found in the upper band. This upper band is shown in electron micrographs to be composed chiefly of closed vesicles, while the particles in the lower band appear morphologically similar to the original ghosts. Total phospholipid phosphorus and cholesterol in the vesicles are enriched to the same extent, giving a lipid to protein ratio of 2 times that found for whole ghosts. The vesicles contain the same individual phospholipids as the ghosts. The protein composition of these vesicles is unique, in that they are almost depleted in the known extrinsic membrane proteins, while containing practically all types of the various glycoproteins of the original membrane. The two main intrinsic membrane proteins (with apparent molecular weights of 160,000 and 100,000) are found almost exclusively in the vesicles, virtually depleted in the residual ghost-like particles. The protein with 160,000 molecular weight is shown here to be a glycoprotein, giving an anomalous molecular weight on sodium dodecyl sulfate gels and having a molecular weight of approximately 50,000 after lipid extraction. This same glycoprotein appears to fractionate with acetylcholinesterase. From the accessibilities of the substrates to the membrane acetylcholinesterase and NADH-diaphorase, it is concluded that the vesicles are right-side-out and sealed to small molecules. There are more membrane sialic acid residues accessible to neuraminidase in the vesicles (in terms of number of residues/mg og membrane protein) than in ghosts, further supporting the conclustion that these vesicles have a normal orientation and are enriched in glycoproteins. The specific activity of acetylcholinesterase in the vesicles is increased 5- to 6-fold over that found in the original ghosts and almost 20-fold over that in the residual ghost-like particles. Consequently, spontaneous vesiculation occurs simultaneously with the enrichement of specific membrane proteins in certain regions of the lipid bilayer. It is postulated that these domains in the membrane, containing clusters of specific intrinsic membrane proteins, bud out and subsequently release glycoprotein-enriched lipid vesicles.

Hormone receptor mobility and catecholamine binding in membranes. A theoretical model.

[3H]-Catecholamine binding to intact cells, isolated cell membranes, and to several isolated macromolecules has been shown by several laboratories to be neither stereospecific nor inhibited by known beta-antagonists. Since additional evidence indicates that this binding is not an artifact (i.e. due neither to the binding of a catecholamine oxidation product nor hormone binding to a catabolic enzyme such as COMT), the question remains as to whether this represents binding to a bona fide membrane receptor. Because all ligands which bind strongly or compete for this binding possess a catechol group, one possible explanation is that the binding affinity is primarily determined by the catechol moiety, whereas the correct stereoisomer of the side chain is necessary to activate the receptor. Thus, although binding is a necessary condition for hormone action, the necessary and sufficient condition for activation of adenyl cyclase is both the catechol group and the correct stereoisomer of the side chain. A theoretical model is developed here to provide a quantitative basis for this hypothesis. This model extends the current concept of distinct subunits in the adenyl cyclase system by separating the receptors from the catalytic sites and placing them at separate locations within the membrane. Utilizing the spare receptor model of Furchgott, and the mobility of macromolecules within a "lipid sea," the appropriate equations to predict both hormone binding and enzyme activation are derived. Using the observed affinity constants from catecholamine binding studies, it is then shown that this model can predict the experimental observation and hence explain the apparent dichotomy arising from binding enzyme activation studies.

ATTRIBUTES OF CHLOROCOCCUM SPECIES: A NUMERICAL ANALYSIS(2).

From previously published data, 73 characteristics of 17 species of Chlorococcum were compiled. Comparisons of character states of each character were made, and simple matching coefficients were calculated for each species by following the procedure of Sokal & Michener. From a data matrix of the matching coefficients, a phenogram was constructed according to the unweighted pair group method of Sokal & Sneath. Arithmetic averages were used in transferring data from one matrix to another. The frequency of each character state was calculated, and the character states of the highest frequency (the modes) were used to describe a "typical" Chlorococcum species. Comparisons were made between the "typical" Chlorococcum species and each of the 17 species studied. Simple matching coefficients were also calculated from these comparisons. All of the species had a relatively high affinity for the data of the calculated "typical". Suggestions are made about the use of a "typical" taxonomic unit, and questions are raised about the taxonomic relationship among species of the genus Tetracystis and the genus Chlorococcum.