Acute effects of filipin on the plasmic, hepatic, and biliary cholesterol of the rat.

Twenty-one male Wistar rats, 13 weeks old, were fed ad libitum hyperlipidic diets (28% fats) loaded with cholesterol (1.2%) for 5 weeks. One group of 11 rats was fed saturated fats (diet group "S") and another group of 10 rats was fed polyunsaturated fats (diet group "PU"). On the day they were sacrificed 10 of the rats were injected intravenously with 1 mg of filipin. Contrary to the rats in diet group "PU," the rats in diet group "S" treated with filipin presented certain characteristics that were not found in the nontreated group: They provided evidence of biliary cholestasis accompanied by a decline in the level of secretion of bile salts and phospholipids into bile. The concentrations of both free and esterified cholesterol in plasma fell and the amount of (esterified) hepatic cholesterol rose, although there was no change due to the filipin in the amounts of hepatic phospholipids. Explanatory hypotheses for these phenomena were considered, first, at the site of plasma membranes where filipin binds selectively to the cholesterol in the membrane, causing a disruption which probably disturbs the absorbance of circulating lipoproteins, especially that of hepatocyte cells, particularly in diet group "PU." Second, the effects of filipin on subcellular membranes seem to disturb the secretion of lipids and lipoproteins into bile and plasma, especially in diet group "S." Last, at the intracellular level, filipin appears to have a blocking effect on the organelles involved in biliary lipid secretion. The activity of certain enzymes such as cholesterol esterase may also be blocked, particularly in diet group "S," which would explain the accumulation of esterified cholesterol in liver.

Influence of acute injection of chloroquine on the biliary secretion of lipids and lysosomal enzyme on rats.

Phospholipids and cholesterol combine with a protein fraction (IgA and an acid polypeptide) in bile to form the bile lipoprotein complex. We wished to determine whether lysosomes participated only on IgA secretion or if their secretory role also involved the lipid components of the bile complex. This aspect was studied with a single acute injection of chloroquine, a lysosomotropic drug. The results show that a nonnegligible quantity of IgA travels through the lysosomes. In addition, phospholipid and cholesterol levels undergo a significant (P less than 0.05) decrease 1 hr after injection before increasing to normal levels. In contrast to the total inhibition of protein secretion (beta-glucuronidase, acid phosphatase), a transitory decrease of the secretion of bile lipids takes place that suggest secretory mechanisms involving organelles other than lysosomes.

Adsorption of pancreatic (pro)phospholipase A2 to various physiological substrates.

The adsorption of pancreatic phospholipase was studied in vitro in the presence of egg yolk lipoprotein emulsion, Intralipid emulsion, and milk fat globules. When the emulsions are incubated with bile salts, the latter dissociate a considerable fraction of the phospholipids initially associated with the emulsions, leading to the coexistence of an emulsified phase and a phase of mixed micelles. After the addition of pancreatic phospholipase A2, gel filtration shows that the enzyme was more than 90% bound to mixed micelles, regardless of the type of emulsion used. Comparable results were obtained by replacing the bile salts with human gallbladder bile. In parallel, pancreatic zymogen was never found to be bound to any of the lipid structures present (emulsion or mixed micelles). When the catalytic site of pancreatic phospholipase A2 was blocked with 4-bromophenacylbromide, there was no fixation on mixed micelles. Fixation was restored by the presence of lysolecithins and fatty acids in the incubation mixtures. The partial transformation of all emulsified substrates to mixed micelles by bile salts in vivo would thus lead to optimum activity of pancreatic phospholipase A2.

Immunohistochemical localization of the apoproteins of the bile lipoprotein complex in the human intestine.

The human bile lipoprotein complex includes an apoprotein fraction of IgA fragments and an acidic polypeptide tightly bound to bile cholesterol and phosphatidylcholines. The fate of the intestinal bile lipoprotein complex was immunohistochemically studied. Its localization in the human duodenum is consistent with selective capture or synthesis. The result of both might be the presence of bile apoprotein in the bloodstream. These two mechanisms may explain the cross-reaction between the lipoprotein complex apoprotein and the high density lipoprotein.

Comparative immunohistochemical localization of IgA and of the IgA part of the bile lipoprotein complex in dog and rat liver.

In bile, phospholipids and cholesterol were found in close association with an apoprotein fraction which cross-reacted to antiserum anti-IgA. In order to determine the origin of the IgA part of the apo bile lipoprotein complex and to establish its relationship with IgA, th localizations of these two components in rat and in dog livers were compared. Two immunohistochemical methods were used. Whereas IgA was found on the sinusoidal surface of the hepatocytes, in cytoplasmic particles and in bile canaliculi, the IgA part of the apo bile lipoprotein complex was only detected in the bile canaliculi, and in particles which presented the same pattern of location as lysosomes. These results suggest that the IgA part of the apo bile lipoprotein complex could originate from the transit of a part of the internalized IgA via the lysosomal compartment of the hepatocytes.

Pancreatic phospholipase A2 hydrolysis of phosphatidylcholines in various physicochemical states.

Bile salts-phosphatidylcholines-cholesterol mixed micelles, native bile, egg yolk and intralipid emulsions were used as pancreatic phospholipase A2 (EC 3.1.1.4) substrates. The enzyme activity depends on the bile salt/phosphatidylcholine molar ratio. The enzyme had a low specific activity on bile phosphatidylcholines, because of the existence in native bile of a high bile salt/phosphatidylcholine molar ratio generating unfavorable conditions of hydrolysis, as demonstrated with mixed micelles. However when the bile salt/phosphatidylcholine molar ratio from bile was decreased to 2 : 1, enzyme activity increases up to an optimum. This optimal activity was about one third that observed when the substrate was mixed micelles. Under these optimal conditions a simultaneous hydrolysis of intralipid and bile phosphatidylcholine mixture shows comparable initial hydrolysis rates. During an extended incubation, however, nearly all intralipid phosphatidylcholines and only half the bile phosphatidylcholines were hydrolyzed by pancreatic phospholipase A2. Bile salts mixture or native bile desorb a portion of the phosphatidylcholines from the intralipid emulsion in optimally hydrolysable bile salts phosphatidylcholines mixed micelles. These micelles bind about 85% of the enzyme indicating that hydrolysis occurs primarily in the micellar phase. These results are discussed in terms of fat lipolysis in vivo.

The influence of bile salts and bile lipoprotein complex on pancreatic lipase hydrolysis of monomolecular films.

We report a new technique which allows us to follow the lipolysis of monomolecular films in the presence of bile salts by using a 'zero-order' trough (Verger, R. and de Haas, G.H. (1973) Chem. Phys. Lipids 10, 127). The effects of bile salts, the bile lipoprotein complex and colipase on pancreatic lipase hydrolysis of rac-1,2-didodecanoylglycerol films were studied at different surface pressures. Taking into account previous studies, lipase activity was interpreted as a function of its degree of binding to the bile lipoprotein complex.