Zenaida Gatmaitan

Studies of Y and Z, two hepatic cytoplasmic organic anion-binding proteins: effect of drugs, chemicals, hormones, and cholestasis

The process by which various anions, including bilirubin and several dyes, drugs, hormones and their metabolites, are transferred from plasma into the liver cell is poorly understood. Two hepatic cytoplasmic proteins, Y and Z, that bind various organic anions in vivo and in vitro have been postulated to be involved in this process. The concentration of Y, the major organic anion-binding protein, increases in rat liver after administration of phenobarbital in association with enhanced organic anion transfer from plasma into liver as determined by initial plasma disappearance rate (K(1)) and hepatic dye content for sulfobromophthalein (BSP) and indocyanine green (ICG), as well as increased relative hepatic storage of BSP. Acute bile duct ligation failed to alter plasma disappearance or hepatic content of BSP in normal or phenobarbital-treated rats. Other drugs and chemicals which cause proliferation of hepatic smooth endoplasmic reticulum and enhancement of drug metabolism, such as allylisopropylacetamide, dieldrin, DDT, 3-methylcholanthrene, and benzpyrene increased Y and BSP K(1) and, where studied, hepatic BSP content. Alcohol feeding had no effect on Y, Z, or K(1) for BSP. Hypophysectomy and thyroidectomy increased Y but decreased K(1) and, where studied, hepatic content of BSP. Of several hormones studied, only thyroxine restored Y and K(1) to normal in hypophysectomized or thyroidectomized rats. Mice with congenital pituitary insufficiency also manifested increased Y which returned to normal after thyroxine administration. In hormone-deficient rats and mice, phenobarbital administration produced a further increase in Y suggesting that different mechanisms may be responsible for the change in Y resulting from drug administration and hormonal deprivation. Thyroxine, testosterone, or hydrocortisone did not alter BSP K(1) or Y in normal rats.Cholestasis produced by ethinyl estradiol administration or biliary obstruction reduced Y, Z, BSP K(1) and hepatic BSP content. These results support the hypothesis that Y and Z are involved in the transfer of BSP, ICG, and possibly other organic anions from plasma into the liver. The concentration of Y increased after administration of various drugs and chemicals as well as in thyroid deficiency. Thyroid hormone appears to be important in regulation of the intracellular concentration of Y. Because thyroid deficiency increased Y but decreased BSP K(1) and hepatic BSP content, other factors beside Y and Z influence hepatic organic anion uptake.

The Function of Gp170, the Multidrug- Resistance Gene Product, in the Brush Border of Rat Intestinal Mucosa

Gp170 is a transmembrane glycoprotein that is overexpressed in multidrug-resistant tumor cells and is present in the apical plasma membrane domain of small intestinal mucosal cells. The function of Gp170 was studied in the small intestine of the rat. Jejunal and ileal brush border membrane vesicles, but not basolateral membrane vesicles, manifested adenosine triphosphate (ATP)-dependent transport of daunomycin, a substrate for Gp170, and contained a approximately 170-kilodalton protein that reacts with anti-Gp170 monoclonal antibody. Whereas ATP supported daunomycin transport, nonhydrolyzable ATP analogues were ineffective. ATP-dependent daunomycin transport by brush border vesicles was unidirectional (inside to outside) and temperature dependent and was blocked by Gp170 inhibitors but not by taurocholate or bromsulphalein glutathione. Studies using everted small intestine revealed transport of rhodamine 123, a Gp170 substrate, from the serosal surface through the mucosa and inhibition by Gp170 inhibitors. These results suggest that Gp170 in rat small intestinal brush border membrane vesicles is an ATP-dependent efflux pump responsible for the transport of Gp170 substrates into the small intestinal lumen. Gp170 may protect against exogenously derived potentially damaging hydrophobic cations and contribute to the rarity of small intestinal cancer in humans and many animals.

The Role of Phosphoinositide 3-Kinase in Taurocholate-induced Trafficking of ATP-dependent Canalicular Transporters in Rat Liver

Recent studies indicate that wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase, interferes with bile acid secretion in rat liver; taurocholate induces recruitment of ATP-dependent transporters to the bile canalicular membrane, and PI 3-kinase products are important in intracellular trafficking. We investigated the role of PI 3-kinase in bile acid secretion by studying the in vivo effect of taurocholate, colchicine, and wortmannin on bile acid secretion, kinase activity, and protein levels in canalicular membrane vesicle (CMV) and sinusoidal membrane vesicle (SMV) fractions from rat liver. Treatment of rats or perfusion of isolated liver with taurocholate significantly increased PI 3-kinase activity in both membrane fractions. Taurocholate increased protein content of ATP-dependent transporters, which were detected only in CMVs, whereas increased levels of p85 and a cell adhesion molecule, cCAM 105, were observed in both fractions. Colchicine prevented taurocholate-induced changes in all proteins studied, as well as the increase in PI 3-kinase activity in CMVs, but it resulted in further accumulation of PI 3-kinase activity, p85, and cCAM 105 in SMVs. These results indicate that taurocholate-mediated changes involve a microtubular system. Wortmannin blocked taurocholate-induced bile acid secretion. The effect was more profound when wortmannin was administered prior to treatment with taurocholate. When wortmannin was given after taurocholate, the protein levels of each ATP-dependent transporter were maintained in CMVs, whereas the levels of p85 and cCAM decreased in both membrane fractions. Perfusion of liver with wortmannin before taurocholate administration blocked accumulation of all proteins studied in CMVs and SMVs. These results indicate that PI 3-kinase is required for intracellular trafficking of itself, as well as of ATP-dependent canalicular transporters.

Structure and Function of P-Glycoprotein in Normal Liver and Small Intestine

Publisher Summary This chapter discusses the structure and function of P-glycoprotein in normal liver and small intestine. Mammalian cells selected for resistance to a single cytotoxic agent display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. This is the “multidrug resistance” (MDR) phenomenon. The emergence of multidrug resistance in cultured cells is associated with the overexpression of a membrane glycoprotein called “P-glycoprotein” (or Gp170). P-Glycoprotein appears to function as an ATP-dependent transporter that pumps drugs out of cells. It interacts with a diverse group of substrates most of which are hydrophobic lipid-soluble organic cations of natural origin including anthracyclines, vinca alkaloids, and colchicines. Based on the sequence homology between P-glycoprotein and known transport proteins, a model for P-glycoprotein has been proposed in which P-glycoprotein is responsible for ATP-dependent efflux of drugs through a plasma membrane channel that is formed by transmembrane domains of one or more P-glycoprotein molecules. Energy is presumably derived from ATP hydrolysis using ATP bound to one or both nucleotide binding sites. P-glycoprotein levels in drug-resistant cells correlate with the amount of drug transported out of the cell, but whether the initial rate of efflux is affected is less established.

Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR-) with inherited conjugated hyperbilirubinemia.

Bilirubin is conjugated with glucuronic acid in hepatocytes and subsequently secreted in bile. The major conjugate is bilirubin diglucuronide. Using sealed vesicles which are primarily derived from the canalicular (CMV) and sinusoidal (SMV) membrane vesicle domains of the plasma membrane of hepatocytes, we demonstrated that bilirubin glucuronides are transported by CMV by both ATP- and membrane potential-dependent transport systems. In CMV from normal rats, these processes are additive. In CMV from TR- rats, which have an autosomal recessively inherited defect in biliary secretion of nonbile acid organic anions, ATP-dependent transport of bilirubin diglucuronide was absent whereas the membrane potential driven system was retained. Other canalicular ATP-dependent transport systems, which were previously described for organic cations and bile acids, are functionally retained in TR- rats. Our study indicates that bilirubin glucuronides are primarily secreted into the bile canaliculus by an ATP-dependent mechanism which is defective in an animal model of the human Dubin-Johnson syndrome.

Serotonin stimulates a Ca2+ permanent nonspecific cation channel in hepatic endothelial cells

The contraction of hepatic endothelial cell fenestrae after exposure to serotonin is associated with an increase in intracellular Ca2+ which is derived from extracellular Ca2+, is inhibited by pertussis toxin and is not associated with activation of phosphoinositol turnover or cAMP. Using cell-attached and excised patches in primary cultures of rat hepatic endothelial cells, we identified a serotonin-activated channel with conductance of 26.4+/-2.3 pS. The channel was also permeant to Na+, K+ and Ca++ but not to anions. In cell-attached patch recordings, addition of serotonin to the bath significantly increased channel activity with Ca2+ or Na+ as the charge-carrying ions. This channel provides a mechanism whereby serotonin can raise the cytosolic Ca2+ concentration in hepatic endothelial cells.

Ligandin reverses bilirubin inhibition of liver mitochondrial respiration in vitro.

Extract: Ligandin, an abundant cytoplasmic binding protein of bilirubin and other ligands in liver cells, completely prevented the inhibitory effect of bilirubin on respiration and oxidative phosphorylation by isolated rat liver mitochondria. At equimolar concentrations of bilirubin and ligandin or human serum albumin, mitochondrial respiration was fully restored to control values. At greater ratios of bilirubin and ligandin or human serum albumin, the latter had a stronger protective effect. These studies suggest that ligandin may have a physiologic role in protecting mitochondrial systems against bilirubin toxicity.Speculation: Some organs, such as liver, kidney, and intestine, transport various organic anions such as bilirubin and possess a soluble binding protein (ligandin) for protection of intracellular organelles. Other organs, such as the brain, lack this protective mechanism and, therefore, are more susceptible to toxicity as, for example, from bilirubin (kernicterus).