Colchicine, a drug which interferes with microtubular function, has no effect on the secretion of taurodehydrocholate into bile; it is therefore suggested that bile salts are unlikely to be packaged in vesicles during cellular transit from sinusoidal to canalicular membranes. Colchicine greatly reduces the secretion of phospholipid and cholesterol into bile; it is suggested that this is due to an interruption in the supply of vesicles bringing lipids to repair the canalicular membrane during bile salt output. In the absence of the protective effect of a continuous supply of repair vesicles, micelleforming bile salts damage the canalicular membrane; the increased concentration of plasma membrane enzymes in bile and the increased aspartate aminotransferase activity in plasma and bile are evidence of this damage. Damage to the canalicular membrane may also be an explanation for the reduction in taurocholate transport and the taurocholate-induced cholestasis which are seen with colchicine-treated livers. Such membrane damage is not observed in colchicine-treated livers during the secretion of the non-micelle forming bile salt, taurodehydrocholate.

Isolated rat livers were perfused under 'one-pass' conditions and bile was collected at 1 min intervals. After 1 min pulse, taurocholate appeared in the collected bile within 2 min, peak output occurring 2 min later. In contrast, the increased output of phospholipids and cholesterol was slower, peak output occurring 6-11 min after the original pulse of taurocholate. These results suggest that mixed micelles cannot be formed inside the cell or during passage of bile salts through the membrane, since bile salt and lipids should then parallel each other. The bile salts must therefore be pumped into the lumen and the lipids added subsequently, due to the actions of the bile salts in the canalicular lumen. It is suggested that the biliary lipid is obtained from microdomains of biliary-type lipid in the canaliculus membrane, which are vesiculated and solubilized by the action of bile salts. It is also suggested that this biliary-type lipid is brought continuously to the membrane via vesicle traffic; this traffic is increased during increased bile-salt output, and is a process that can be inhibited by colchicine.

The pathways for the entry of horseradish peroxidase (HRP) into bile have been investigated using the isolated perfused rat liver operating under one-pass conditions. Following a 1 min one-pass infusion of HRP, two peaks of HRP activity were noted in the bile. The first, at 5-7 min post-infusion, correlated with the biliary secretion of the [3H]methoxyinulin which was infused simultaneously with the HRP. The second peak of HRP activity occurred at 20-25 min, and correlated with the biliary secretion of 125I-IgA, which was also infused simultaneously with the HRP. If the isolated livers were perfused with a medium containing 2.5 microM-colchicine, the biliary secretion of IgA and the second secretion peak of HRP were inhibited by 60%. If rats were pretreated for 12h with alpha-naphthylisothiocyanate (25mg/100g body wt.) prior to liver isolation, the biliary secretion of [3H]methoxyinulin and the first secretion peak of HRP were increased. Taken together, these results suggest that HRP enters the bile via two routes. The faster route, which was increased by alpha-naphthylisothiocyanate and correlated with [3H]methoxyinulin entry into bile, was probably paracellular, involving diffusion across tight junctions. The slower route, which was inhibited by colchicine and correlated with the secretion of IgA, was probably due to transcytosis, possibly within IgA and other transport vesicles.

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.

The factors modulating the maximum secretory rate of cholic acid were investigated. Rats were infused intravenously with cholic acid in measured stepwise increasing doses (1, 2, 3, and 4 mumol X min-1 X 100 g body wt-1). Each dose was infused for 30 min and bile samples were collected every 10 min. Bile flow, bile acid, cholesterol, individual biliary phospholipids, and the fatty acid profiles of the biliary phospholipids were determined. Microsomal and bile canalicular membrane-enriched fractions were isolated from cholic acid-treated rats at the end of the experiment. Membranes were analyzed for cholesterol, phospholipid, and phospholipid fatty acid composition. During cholic acid infusion, the secretion rates of bile acid, cholesterol, phospholipid, and bile flow initially increased and then declined. No evidence of liver cell damage was observed by light or electron microscopy. Maximum phospholipid secretion rate (13.5 nmol X min-1 X g-1) occurred before peak bile flow and bile acid secretory rate maximum (4.72 microliter X min-1 X g-1 and 375 nmol X min-1 X g-1). When phospholipid output declined, the proportion of sphingomyelins and phosphatidylethanolamine relative to phosphatidylcholine increased. This was also reflected in the fatty acid composition. Cholic acid infusion caused a decline in microsomal and bile canalicular membrane phospholipid content without affecting their phospholipid composition. Depletion of membrane phospholipid resulted in an increase in the cholesterol:phospholipid ratio, which is suggested to be the underlying mechanism for modulating cholic acid secretion.