Medical Physiology, 3rd Edition

Uptake, Processing, and Secretion of Compounds by Hepatocytes

The liver metabolizes an enormous variety of compounds that are brought to it by the portal and systemic circulations. These compounds include endogenous molecules (e.g., bile salts and bilirubin, which are key ingredients of bile) and exogenous molecules (e.g., drugs and toxins). The hepatocyte handles these molecules in four major steps (Fig. 46-5A): (1) the hepatocyte imports the compound from the blood across its basolateral (i.e., sinusoidal) membrane, (2) the hepatocyte transports the material within the cell, (3) the hepatocyte may chemically modify or degrade the compound intracellularly, and (4) the hepatocyte excretes the molecule or its product or products into the bile across the apical (i.e., canalicular) membrane. Thus, compounds are secreted in a vectorial manner through the hepatocyte.

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FIGURE 46-5 Transporters in hepatocyte. A, The hepatocyte can process compounds in four steps: (1) uptake from blood across the basolateral (i.e., sinusoidal) membrane; (2) transport within the cell; (3) control chemical modification or degradation; and (4) export into the bile across the apical (i.e., canalicular) membrane. B, The hepatocyte has a full complement of housekeeping transporters. C, Bile acids can enter the hepatocyte in any of several forms: the unconjugated salt (BA); the neutral, protonated bile acid (H ⋅ BA); or the bile salt conjugated to taurine or glycine (BA-Z, where Z represents taurine or glycine). The three pathways for bile acid entry across the basolateral membrane are the Na+-driven transporter NTCP, which prefers BA-Z but also carries BA; nonionic diffusion of H ⋅ BA; and an OATP. Binding proteins (BPs) may ferry conjugated bile acids across the cytoplasm. Some bile acids are conjugated to sulfate or glucuronate (Y); these exit the cell across the canalicular membrane via the MRP2 (multidrug resistance–associated protein 2) transporter. Most bile acids are conjugated to glycine or taurine (Z) prior to their extrusion into the bile via BSEP. D, Organic anions (OA), including bile acids and bilirubin, may enter across the basolateral membrane via an OATP. After conjugation with sulfate or glucuronate (Y), these compounds may be extruded into the bile by MRP2. GSH synthesized in the hepatocyte, after conjugation to Y, can enter the canaliculus via MRP2. Unconjugated GSH can enter the canaliculus via an unidentified transporter. GSH can exit the hepatocyte across the basolateral membrane via an OATP. AA, amino acid.

An Na-K pump at the basolateral membranes of hepatocytes provides the energy for transporting a wide variety of solutes via channels and transporters

Like other epithelial cells, the hepatocyte is endowed with a host of transporters that are necessary for basic housekeeping functions. imageN46-2 To the extent that these transporters are restricted to either the apical or basolateral membrane, they have the potential of participating in net transepithelial transport. For example, the Na-K pump (see pp. 115–117) at the basolateral membrane of hepatocytes maintains a low [Na+]i and high [K+]i (see Fig. 46-5B). A basolateral Ca pump (see p. 118) maintains [Ca2+]i at an extremely low level, ~100 nM, as in other cells. The hepatocyte uses the inwardly directed Na+ gradient to fuel numerous active transporters, such as the Na-H exchanger, Na/HCO3 cotransporter, and Na+-driven amino-acid transporters. As discussed below, the Na+ gradient also drives one of the bile acid transporters. The hepatocyte takes up glucose via the GLUT2 facilitated-diffusion mechanism (see p. 114), which is insensitive to regulation by insulin.

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Hepatocyte Housekeeping Functions

Contributed by Fred Suchy

As noted in the text, the basolateral membrane of the hepatocyte has both K+ and Cl channels. The basolateral K+ conductance is high and is regulated by cAMP, [Ca2+]i, cell volume, and temperature. The basolateral Cl conductance is under the regulation of hormones and cell volume.

As is the case for most cells, hepatocytes actively regulate their intracellular pH (see pp. 644–645) using two acid extruders, the basolateral (i.e., sinusoidal) Na-H exchanger and an electrogenic Na/HCO3cotransporter. The apical (i.e., canalicular) Cl-HCO3 exchanger may contribute as an acid loader. The pH gradient across the canalicular membrane also drives the transport of inorganic solutes (e.g., HCO3-SO4 exchange) and maintains the transmembrane gradients of weak acids and bases that cross the membrane by nonionic diffusion (see p. 784).

The basolateral membrane has both K+ and Cl channels. The resting membrane potential (Vm) of −30 to −40 mV is considerably more positive than the equilibrium potential for K+ (EK) because of the presence of numerous “leak” pathways, such as the aforementioned electrogenic Na+-driven transporters as well as Cl channels (ECl = Vm).

Hepatocytes take up bile acids, other organic anions, and organic cations across their basolateral (sinusoidal) membranes

Bile Acids and Salts

The primary bile acids are cholic acid and chenodeoxycholic acid, both of which are synthesized by hepatocytes (see p. 959, below). Other “secondary” bile acids form in the intestinal tract as bacteria dehydroxylate the primary bile acids. Because the pK values of the primary bile acids are near neutrality, most of the bile acid molecules are neutral; that is, they are bile acids (H ⋅ BA) and thus are not very water soluble. Of course, some of these molecules are deprotonated and hence are bile salts (BA). The liver may conjugate the primary bile acids and salts to glycine or taurine (Z in Fig. 46-5C), as well as to sulfate or glucuronate (Y in Fig. 46-5C). Most of the bile acids that the liver secretes into the bile are conjugated, such as taurocholate (the result of conjugating cholic acid to taurine). These conjugated derivatives have a negative charge and hence they, too, are bile salts (BA-Z and BA-Y). Bile salts are far more water soluble than the corresponding bile acids.

Because the small intestine absorbs some bile acids and salts, they appear in the blood plasma, mainly bound to albumin, and are presented to the hepatocytes for re-uptake. This recycling of bile acids, an example of enterohepatic circulation (see p. 962 below). Dissociation from albumin occurs before uptake. Surprisingly, the presence of albumin actually stimulates Na+-dependent taurocholate uptake, perhaps by increasing the affinity of the transporter for taurocholate.

Uptake of bile acids has been studied extensively and is mediated predominantly by an Na+-coupled transporter known as Na/taurocholate cotransporting polypeptide or NTCP (a member of the SLC10A1 family; see Fig. 46-5C). This transporter is a 50-kDa glycosylated protein, and it appears to have seven membrane-spanning segments. NTCP handles unconjugated bile acids, but it has a particularly high affinity for conjugated bile acids. In addition, NTCP can also transport other compounds, including neutral steroids (e.g., progesterone, 17β-estradiol sulfate), cyclic oligopeptides (e.g., amantadine and phalloidin), and a wide variety of drugs (e.g., verapamil, furosemide). imageN46-3

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Regulation of Na/Taurocholate Cotransport

Contributed by Emile Boulpaep, Walter Boron

Bile acid uptake via the Na/taurocholate cotransporting polypeptide (NTCP) is under the regulation of several second messengers. For example, cAMP stimulates taurocholate uptake, whereas this effect is blocked by inhibitors of protein kinase A. This direct stimulation presumably reflects the phosphorylation of the transporter or an essential activator. cAMP also stimulates uptake indirectly by increasing translocation of the transport protein to the membrane.

Certain hormones, such as prolactin, also stimulate bile acid uptake directly.

As is the case for many other transporters, NTCP activity is low in the fetus and neonate and increases with development.

NTCP has now been classified as a member of the SLC10 gene family (see Table 5-4) of Na/bile-salt cotransporters. For a detailed discussion of the family members, consult the review by Hagenbuch and Dawson listed below.

Reference

Hagenbuch B, Dawson P. The sodium bile salt cotransport family SLC10. Pflugers Arch. 2004;447:566–570.

Although NTCP also carries unconjugated bile acids, as much as 50% of these unconjugated bile acids may enter the hepatocyte by passive nonionic diffusion (see Fig. 46-5B). Because unconjugated bile acids are weak acids of the form

image (46-1)

the neutral H ⋅ ΒΑ form can diffuse into the cell. Conjugation of bile acids enhances their hydrophilicity (taurine more so than glycine) and promotes dissociation of the proton from the side chain (i.e., lowers the pKa), thus raising the concentration of BA. Both properties decrease the ability of bile acid to traverse membranes via passive nonionic diffusion.

Organic Anions

The organic anion–transporting polypeptides (OATPs) are members of the SLC21 family (see p. 125) imageN46-4 and mediate the Na+-independent uptake of a wide spectrum of endogenous and exogenous amphipathic compounds—including bile acids, bilirubin, eicosanoids, steroid and thyroid hormones, prostaglandins, statin drugs, methotrexate, bromosulfophthalein, and many xenobiotics. Individual OATPs share considerable overlap in substrate specificity and can substantially influence the pharmacokinetics and pharmacological efficacy of drugs they carry. OATPs—predicted to have 12 membrane-spanning segments, with intracellular amino and carboxy termini—appear to exchange organic anions for intracellular image (see Fig. 46-5C, D). Expression of OATPs is under the control, in a cell- and tissue-specific way, of nuclear receptors (FXR, LXR, SXR, CAR; see Table 3-6) and hepatocyte nuclear factor 4 (HNF4). OATP1B1, OATP1B3, and OATP2B1 are liver specific and are located on the sinusoidal (basolateral) membrane of hepatocytes.

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Organic Anion Transporters

Contributed by Emile Boulpaep, Walter Boron

The organic-anion transporting proteins (OATPs) have now been classified as members of the SLC21 gene family (see Table 5-4). For a detailed discussion of the family members, consult the review by Hagenbuch and Meier listed below.

Note that some have inappropriately stated that because this family has an immense number of genes, it really ought to be treated as a superfamily, with the designation SLCO (here the O refers to “organic”). However, the superfamily is large only when one includes genes from all known organisms. The actual number of OATP genes in any given vertebrate organism (humans have 11 such genes) is about the same as for other SLC families. Thus, the OATPs are appropriately described as a “family.”

Reference

Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/SLC21 family: Phylogenetic classification as OATP/SLC0 superfamily, new nomenclature and molecular/functional properties. Pflugers Arch. 2004;447:653–665.

Thus, the basolateral uptake of bile acids into the hepatocyte is a complex process that involves both an Na+-dependent transporter (NTCP) and Na+-independent transporters (OATPs), as well as nonionic diffusion of unconjugated bile acids.

Bilirubin

Senescent erythrocytes are taken up by macrophages in the reticuloendothelial system, where the degradation of hemoglobin leads to the release of bilirubin into the blood (Fig. 46-6A and Box 46-1). The mechanism by which hepatocytes take up unconjugated bilirubin remains controversial. As evidenced by yellow staining of the sclerae and skin in the jaundiced patient, bilirubin can leave the circulation and enter tissues by diffusion. However, uptake of albumin-bound bilirubin by the isolated, perfused rat liver and isolated rat hepatocytes is faster than can occur by diffusion and is consistent with a carrier-mediated process. Electroneutral, electrogenic, and Cl-dependent transport have been proposed (see Fig. 46-6B).

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FIGURE 46-6 Excretion of bilirubin. A, Macrophages phagocytose senescent red blood cells and break the heme down to bilirubin, which travels in the blood, linked to albumin, to the liver. The conversion to the colorless urobilinogen occurs in the terminal ileum and colon, whereas the oxidation to the yellowish urobilin occurs in the urine. B, The hepatocyte takes up bilirubin across its basolateral membrane via an OATP and other unidentified mechanisms. The hepatocyte then conjugates the bilirubin with one or two glucuronic acid residues and exports this conjugated form of bilirubin into the bile. Bacteria in the terminal ileum and colon convert some of this bilirubin glucuronide back to bilirubin. This bilirubin is further converted to the colorless urobilinogen. If it remains in the colon, the compound is further converted to stercobilin, which is the main pigment of feces. If the urobilinogen enters the plasma and is filtered by the kidney, it is converted to urobilin and gives urine its characteristic yellow color. NADP+, oxidized form of nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate.

Box 46-1

Jaundice

Jaundice denotes a yellowish discoloration of body tissues, most notable in the skin and sclera of the eyes. The condition is caused by an accumulation of bilirubin in extracellular fluid, either in free form or after conjugation. Bilirubin is a yellow-green pigment that is the principal degradation product of heme (see Fig. 46-6A), a prosthetic group in several proteins, including hemoglobin.

The metabolism of hemoglobin (Hb) of senescent red cells accounts for 65% to 80% of total bilirubin production. Hb released into the circulation is phagocytized by macrophages throughout the body, which split Hb into globin and heme. Cleavage of the heme ring releases both free iron, which travels in the blood by transferrin, and a straight chain of 4-pyrrole nuclei called biliverdin (see Fig. 46-6A), which the cell rapidly reduces to free bilirubin. This lipophilic form of bilirubin is often referred to as unconjugated bilirubin. After it enters the circulation, unconjugated bilirubin binds reversibly to albumin and travels to the liver, which avidly removes it from the plasma (see Fig. 46-6B). After hepatocytes take up bilirubin, they use UGT1A1 to convert the bilirubin to monoglucuronide and diglucuronide conjugates. These two forms of water-soluble, conjugated bilirubin—which make up the direct bilirubin measured in clinical laboratories—enter the bile canaliculus via MRP2 (see Table 5-6). Although suitable for excretion into bile, conjugated bilirubin cannot be absorbed by the biliary or intestinal epithelia.

Because of avid extraction and conjugation of bilirubin by the liver, the normal plasma concentration of bilirubin, which is mostly of the unconjugated variety, is ~0.5 mg/dL or lower. The skin or eyes may begin to appear jaundiced when the bilirubin level rises to 1.5 to 3 mg/dL.

Jaundice occurs under several circumstances. Increased destruction of red blood cells or hemolysis may cause unconjugated hyperbilirubinemia. Transient physiological neonatal jaundice results from an increased turnover of red blood cells combined with the immaturity of the pathways for conjugation of bilirubin (exacerbated in premature infants) Pathological conditions that can increase bilirubin production in neonates include isoimmunization, heritable hemolytic disorders, and extravasated blood (e.g., from bruises and cephalhematomas). Genetic disorders of bilirubin conjugation include the mild deficiency of UGT1A1 seen in the common Gilbert syndrome and the near-complete or complete deficiency of UGT1A1 seen in the rare Crigler-Najjar syndrome. Extreme unconjugated hyperbilirubinemia can lead to a form of brain damage called kernicterus (from the Dutch kern [nucleus, as in a brain nucleus] + the Greek icteros [jaundice]). Neonatal hyperbilirubinemia is often treated with phototherapy, which converts bilirubin to photoisomers and colorless oxidation products that are less lipophilic than bilirubin and do not require hepatic conjugation for excretion. Photoisomers are excreted mainly in the bile, and oxidation products, predominantly in the urine.

Jaundice can also result from defects in the secretion of conjugated bilirubin from hepatocytes into bile canaliculi (as with certain types of liver damage) or from defects in transiting the bilirubin to the small intestine (as with obstruction of the bile ducts). In either case, conjugated bilirubin refluxes back into the systemic circulation, where it now accounts for most of the bilirubin in plasma. Because the kidneys can filter the highly soluble conjugated bilirubin—in contrast to the poorly soluble free form of bilirubin mostly bound to albumin—it appears in the urine. Thus, in obstructive jaundice, conjugated bilirubin imparts a dark yellow color to the urine. Measurement of free and conjugated bilirubin in serum serves as a sensitive test for detecting liver disease.

Under normal conditions, approximately half of the bilirubin reaching the intestinal lumen is metabolized by bacteria into the colorless urobilinogen (see Fig. 46-6A). The intestinal mucosa reabsorbs ~20% of this soluble compound into the portal circulation. The liver then extracts most of the urobilinogen and re-excretes it into the gastrointestinal tract. The kidneys excrete a small fraction (~20% of daily urobilinogen production) into the urine. Urobilinogen may be detected in urine by using a clinical dipstick test. Oxidation of urobilinogen yields urobilin, which gives urine its yellow color. In the feces, metabolism of urobilinogen yields stercobilin, which contributes to the color of feces. In obstructive jaundice, no bilirubin reaches the intestine for conversion into urobilinogen, and therefore no urobilinogen appears in the blood for excretion by the kidney. As a result, tests for urobilinogen in urine are negative in obstructive jaundice. Because of the lack of stercobilin and other bile pigments in obstructive jaundice, the stool becomes clay colored.

OATP1B1 and OATP1B3 can transport conjugated, and possibly unconjugated, bilirubin in vitro. Indeed, human mutations resulting in the complete deficiency of OATP1B1 or OATP1B3 cause Rotor syndrome, a relatively benign autosomal recessive disorder characterized by conjugated—not unconjugated—hyperbilirubinemia. How did this conjugated bilirubin—made only in hepatocytes—get into the blood? It is now clear that hepatocytes secrete substantial amounts of glucuronidated bilirubin across the sinusoidal membrane into the space of Disse and that OATP1B1/OATP1B3 is responsible for the reuptake of this conjugated bilirubin under physiological conditions. Other hepatic mechanisms may mediate the uptake of unconjugated bilirubin.

Organic Cations

The major organic cations transported by the liver are aromatic and aliphatic amines, including important drugs such as cholinergics, local anesthetics, and antibiotics, as well as endogenous solutes such as choline, thiamine, and nicotinamide (Fig. 46-7). At physiological pH, ~40% of drugs are organic cations, in equilibrium with their respective conjugate weak bases (see p. 628). Members of the organic cation transporter (OCT) family mediate the uptake of a variety of structurally diverse lipophilic organic cations of endogenous or xenobiotic origin (see p. 115). OCT-mediated transport is electrogenic, independent of an Na+ ion or proton gradient, and may occur in either direction across the plasma membrane. Human hepatocytes express only OCT1 (SLC22A1) and OCT3 (SLC22A3), localized to the sinusoidal membrane. OCT1 and OCT3 have partly overlapping substrate specificities. OCT1 is also present in the plasma membrane of cholangiocytes. Acyclovir and lidocaine are examples of OCT1 substrates. The neurotransmitters epinephrine, norepinephrine, and histamine are exclusive OCT3 substrates. In addition to the OCTs, members of the OATP family as well as an electroneutral proton-cation exchanger may contribute to organic cation uptake across the basolateral membrane.

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FIGURE 46-7 Excretion of organic cations and lipids. APL, aminophospholipid; C, cholesterol; PL, phospholipid.

Neutral Organic Compounds

This group of molecules is also taken up by an Na+-independent, energy-dependent process, although the nature of the driving force is not known. The best-characterized substrate is ouabain, uptake of which is inhibited by other neutral steroids, such as cortisol, aldosterone, estradiol, and testosterone. OATP1B1 transports some of these compounds.

We return to Figures 46-5 through 46-7 below, when we discuss the movement of solutes into the bile canaliculus.

Inside the hepatocyte, the basolateral-to-apical movement of many compounds occurs by protein-bound or vesicular routes

Bile Salts

Some compounds traverse the cell while bound to intracellular “binding” proteins (see Fig. 46-5C). The binding may serve to trap the molecule within the cell, or it may be involved in intracellular transport. For bile salts, three such proteins have been identified. In humans, the main bile acid–binding protein appears to be the hepatic dihydrodiol dehydrogenase, one of a large family of dehydrogenases, the catalytic and binding properties of which are organ and species specific. The two others are glutathione-S-transferase B and fatty acid–binding protein. Intracellular sequestration of bile salts by these proteins may serve an important role in bile acid transport or regulation of bile acid synthesis. Transcellular diffusion of bile salts bound to proteins can be detected within seconds after bile salts are applied to hepatocytes; this mechanism may be the primary mode of cytoplasmic transport under basal conditions. Free, unbound bile acids may also traverse the hepatocyte by rapid diffusion.

At high sinusoidal concentrations, hydrophobic bile acids may partition into membranes of intracellular vesicles. These conditions may also cause increased targeting of the vesicles to the canalicular membrane—that is, transcellular bile acid transport by a vesicular pathway. Whether transcellular transport occurs by protein-binding or vesicular pathways, it is unknown how bile acids are so efficiently targeted to the canalicular membrane for excretion into bile.

Bilirubin

After uptake at the basolateral membrane, unconjugated bilirubin is transported to the endoplasmic reticulum (ER), where it is conjugated to glucuronic acid (see Fig. 46-6). Because the resulting bilirubin glucuronide is markedly hydrophobic, it was thought that intracellular transport was mediated by binding proteins such as glutathione-S-transferase B. However, spontaneous transfer of bilirubin between phospholipid vesicles occurs by rapid movement through the aqueous phase, in the absence of soluble proteins. Thus, direct membrane-to-membrane transfer may be the principal mode of bilirubin transport within the hepatocyte. In addition, the membrane-to-membrane flux of bilirubin is biased toward the membrane with the higher cholesterol/phospholipid ratio. Hence, the inherent gradient for cholesterol from the basolateral membrane to the ER membrane may direct the flux of bilirubin to the ER.

In phase I of the biotransformation of organic anions and other compounds, hepatocytes use mainly cytochrome P-450 enzymes

The liver is responsible for the metabolism and detoxification of many endogenous and exogenous compounds. Some compounds taken up by hepatocytes (e.g., proteins and other ligands) are completely digested within lysosomes. Specific carriers exist for the lysosomal uptake of sialic acid, cysteine, and vitamin B12. Clinical syndromes resulting from an absence of these carriers have also been identified. The lysosomal acid hydrolases cleave sulfates, fatty acids, and sugar moieties from larger molecules.

Hepatocytes handle other compounds by biotransformation reactions that usually occur in three phases. Phase I reactions represent oxidation or reduction reactions in large part catalyzed by the P-450 cytochromes. The diverse array of phase I reactions includes hydroxylation, dealkylation, and dehalogenation, among others. The common feature of all of these reactions is that one atom of oxygen is inserted into the substrate. Hence, these monooxygenases make the substrate (RH) a more polar compound, poised for further modification by a phase II reaction. For example, when the phase I reaction creates a hydroxyl group (ROH), the phase II reaction may increase the water solubility of ROH by conjugating it to a highly hydrophilic compound such as glucuronate, sulfate, or glutathione:

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(46-2)

Finally, in phase III, the conjugated compound moves out of the liver via transporters on the sinusoidal and canalicular membranes.

The P-450 cytochromes are the major enzymes involved in phase I reactions. Cytochromes are colored proteins that contain heme for use in the transfer of electrons. Some cytochromes—not the P-450 system—are essential for the electron transport events that culminate in oxidative phosphorylation in the mitochondria. The P-450 cytochromes, so named because they absorb light at 450 nm when bound to CO, are a diverse but related group of enzymes that reside mainly in the ER and typically catalyze hydroxylation reactions. Fifty-seven human CYP genes encode hundreds of variants of cytochrome P-450 enzymes (see Table 50-2). Genetic polymorphisms exist in the genes encoding all the main P-450 enzymes that contribute to drug and other xenobiotic metabolism, and the distribution and frequency of variant alleles can vary markedly among populations.

In this text, we encounter P-450 oxidases in two sets of organs. In cells that synthesize steroid hormones—the adrenal cortex (see p. 1021), testes (see p. 1097), and ovary (see p. 1117) and placenta (see Table 56-5)—the P-450 oxidases are localized either in the mitochondria or in the ER, where they catalyze various steps in steroidogenesis. In the liver, these enzymes are located in the ER, where they catalyze a vast array of hydroxylation reactions involving the metabolism of drugs and chemical carcinogens, bile acid synthesis, and the activation and inactivation of vitamins. The same reactions occur in other tissues, such as the intestines and the lungs.

Hepatic microsomal P-450 enzymes have similar molecular weights (48 to 56 kDa). The functional protein is a holoenzyme that consists of an apoprotein and a heme prosthetic group. The apoprotein region confers substrate specificity, which differs among the many P-450 enzymes. These substrates include RH moieties that are as wide ranging as the terminal methyl group of fatty acids, carbons in the rings of steroid molecules, complex heterocyclic compounds, and phenobarbital. In general, phase I processes add or expose a functional group, a hydroxyl group in the case of the P-450 oxidases, which renders the molecule reactive with phase II enzymes. The metabolic products of phase I may be directly excreted, but more commonly, because of only a modest increment in solubility, further metabolism by phase II reactions is required.

In phase II of biotransformation, conjugation of phase I products makes them more water soluble for secretion into blood or bile

In phase II, the hepatocyte conjugates the metabolites generated in phase I to produce more hydrophilic compounds, such as glucuronides, sulfates, and mercapturic acids. These phase II products are readily secreted into the blood or bile. Conjugation reactions are generally considered to be the critical step in detoxification. Either a defect in a particular enzyme, which may result from a genetic defect, or saturation of the enzyme with excess substrate may result in a decrease in the overall elimination of a compound. One example is gray syndrome, a potentially fatal condition that occurs after the administration of chloramphenicol to newborns who have low glucuronidation capacity. Infants have an ashen gray appearance and become weak and apathetic, and complete circulatory collapse may ensue.

Hepatocytes use three major conjugation reactions:

1. Conjugation to glucuronate. The uridine diphosphate–glucuronosyltransferases (UGTs), which reside in the smooth endoplasmic reticulum (SER) of the liver, are divided into two families based on their substrate specificity. The UGT1 family consists of at least nine members encoded by genes located on chromosome 2. These UGTs catalyze the conjugation of glucuronic acid with phenols or bilirubin (see Fig. 46-6B). The UGT2 family contains at least nine UGTs encoded by genes on chromosome 4. These UGTs catalyze the glucuronidation of steroids or bile acids. The two members of the UGT3 family reside on chromosome 5. Because UGT1s are essential for the dual conjugation of bilirubin (see Fig. 46-6B) and because only conjugated bilirubin can be excreted in bile, congenital absence of UGT1A1 activity results in jaundice from birth and bilirubin encephalopathy, as seen in patients with Crigler-Najjar syndrome type I.

2. Conjugation to sulfate. The sulfotransferases—which are located in the cytosol rather than in the SER—catalyze the sulfation of steroids, catechols, and foreign compounds such as alcohol and metabolites of carcinogenic hydrocarbons. Their substrate specificity is greater than that of the UGTs. The different cellular localization of these two groups of enzymes suggests that they act cooperatively rather than competitively. In general, sulfates are not toxic and are readily eliminated, with the exception of sulfate esters of certain carcinogens.

3. Conjugation to glutathione. Hepatocytes also conjugate a range of compounds to reduced glutathione (GSH) for excretion and later processing in either the bile ducts or kidney (Fig. 46-8). Glutathione is a tripeptide composed of glutamate γ-linked to cysteine, which, in turn, is α-linked to glycine. The liver has the highest concentration of glutathione (~5 mM), with ~90% found in the cytoplasm and 10% in the mitochondria. Glutathione-S-transferases, which are mainly cytosolic, catalyze the conjugation of certain substrates to the cysteine moiety of GSH. Substrates include the electrophilic metabolites of lipophilic compounds (e.g., epoxides of polycyclic aromatic hydrocarbons), products of lipid peroxidation, and alkyl and aryl halides. In some cases, the conjugates are then secreted into bile and are further modified by removing the glutamyl residue from the glutathione by γ-glutamyl transpeptidase on the bile duct epithelial cell. The fate of glutathione-S-conjugates in bile is largely unknown. Some (e.g., the leukotrienes) undergo enterohepatic circulation. In other cases, the glutathione conjugates are secreted into plasma and are filtered by the kidney, where a γ-glutamyl transpeptidase on the proximal tubule brush border again removes the glutamyl residue. Next, a dipeptidase removes the glycine residue to produce a cysteine-S-conjugate. The cysteine-S-conjugate is either excreted in the urine or is acetylated in the kidney or liver to form a mercapturic acid derivative, which is also excreted in the urine. Although glutathione conjugation is generally considered a detoxification reaction, several such conjugates undergo activation into highly reactive intermediates.

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FIGURE 46-8 Conjugation to GSH and formation of mercapturic acids. The first step is for glutathione-S-transferase to couple the target compound (R) to the S on the cysteine residue of GSH. After MRP2 transports this GSH conjugate into the canalicular lumen (see Fig. 46-5D), a γ-glutamyl transpeptidase may remove the terminal glutamate residue. Alternatively, the conjugate may reach the blood and be filtered by the kidney where a γ-glutamyl transpeptidase at the brush border and a dipeptidase generate a cysteine derivative of R. Acetylation yields the mercapturic acid derivative, which appears in the urine.

Other forms of conjugation include methylation (e.g., catechols, amines, and thiols), acetylation (e.g., amines and hydrazines), and conjugation (e.g., bile acids) with amino acids such as taurine, glycine, or glutamine.

The involvement of multiple enzyme systems in these detoxification reactions facilitates the rapid removal of toxic species and provides alternative pathways in the event of failure of the preferred detoxification mechanism.

In phase III of biotransformation, hepatocytes excrete products of phase I and II into bile or sinusoidal blood

Phase III involves multidrug transporters of the ATP-binding cassette (ABC) family (see Table 5-6)—such as MDR1 (ABCB1), MRP1 (ABCC1), and MRP2 (ABCC2)—located on the canalicular membrane. These transporters have broad substrate specificity and play an important role in protecting tissues from toxic xenobiotics and endogenous metabolites. However, their overexpression often leads to the development of resistance to anticancer drugs and can adversely affect therapy with other drugs, such as antibiotics. MRP2, which transports conjugated bilirubin, can also transport other conjugated substrates, including drugs and xenobiotics conjugated to glutathione.

ABC proteins of broad substrate specificity—such as MRP4 (ABCC4) and MRP6 (ABCC6)—are also expressed on the basolateral or sinusoidal membrane of hepatocytes. MRP4, which has been studied best, facilitates the efflux of bile-salt conjugates (see Fig. 46-5C), conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs into sinusoidal blood. These sinusoidal efflux pumps are upregulated in cholestasis, which enables renal elimination of substances with compromised canalicular transport.

The interactions of xenobiotics with nuclear receptors control phase I, II, and III

The nuclear receptors (see Table 3-6) for xenobiotics, the steroid and xenobiotic receptor (SXR, also known as the pregnane X receptor, or PXR), the constitutive androstane receptor (CAR), and the aryl hydrocarbon receptor (AhR) coordinately induce genes involved in the three phases of xenobiotic biotransformation. Many xenobiotics are ligands for orphan NRs, CAR, and SXR, which heterodimerize with the retinoid X receptor (RXR) and transcriptionally activate the promoters of many genes (see pp. 90–92) involved in drug metabolism. Similarly, many polycyclic aromatic hydrocarbons bind to AhR, which then dimerizes with the AhR nuclear translocator (ARNT), inducing cytochrome P-450 genes.

Enzymes upregulated by SXR include the phase I drug-metabolizing enzymes of the P-450 family, such as CYP3A, which metabolizes >50% of all drugs in humans. SXR also activates the phase II enzyme glutathione-S-transferase, which is critical for catalyzing conjugation of many substrates to glutathione. SXR also upregulates MDR1 (see Table 5-6). Although these pathways are for the most part hepatoprotective, a particular compound may elicit SXR-mediated alterations in CYP3A activity that may profoundly influence the metabolism of another drug—perhaps thereby compromising the therapeutic efficacy of that drug or enhancing the production of a toxic metabolite.

The constitutive androstane receptor (CAR) is also an important regulator of drug metabolism. CAR regulates all the components of bilirubin metabolism, including uptake (possibly through OATP), conjugation (UTG1A1), and excretion (MRP2).

Hepatocytes secrete bile acids, organic anions, organic cations, and lipids across their apical (canalicular) membranes

At the apical membrane, the transport of compounds is generally unidirectional, from cell to canalicular lumen. An exception is certain precious solutes, such as amino acids and adenosine, which are reabsorbed from bile by Na+-dependent secondary active transport systems.

Bile Salts

Bile-salt transport from hepatocyte to canalicular lumen (see Fig. 46-5C) occurs via an ATP-dependent transporter called the bile-salt export pump (BSEP or ABCB11; see Table 5-6). BSEP has a very high affinity for bile salts (taurochenodeoxycholate > taurocholate > tauroursodeoxycholate > glycocholate). The electrical charge of the side chain is an important determinant of canalicular transport inasmuch as only negatively charged bile salts are effectively excreted. Secretion of bile salts occurs against a significant cell-to-canaliculus concentration gradient, which may range from 1 : 100 to 1 : 1000. Mutations in the BSEP gene can, in children, cause a form of progressive intrahepatic cholestasis that is characterized by extremely low bile acid concentrations in the bile.

Organic Anions

Organic anions that are not bile salts move from the cytoplasm of the hepatocyte to the canalicular lumen largely via MRP2 (ABCC2, see Table 5-6 and Fig. 46-5D). MRP2 is electrogenic, ATP dependent, and has a broad substrate specificity imageN46-5 —particularly for divalent, amphipathic, phase II conjugates with glutathione, glucuronide, glucuronate, and sulfates. Its substrates include bilirubin diglucuronide, sulfated bile acids, glucuronidated bile acids, and several xenobiotics. In general, transported substrates must have a hydrophobic core and at least two negative charges separated by a specific distance. MRP2 is critical for the transport of GSH conjugates across the canalicular membrane into bile. Although MRP2 has a low affinity for GSH, functional studies suggest that other mechanisms for GSH transport exist. Animal models of defective MRP2 exhibit conjugated hyperbilirubinemia, which corresponds phenotypically to Dubin-Johnson syndrome in humans. Another canalicular efflux pump for sulfated conjugates is breast cancer resistance protein (BCRP or ABCG2), which transports estrone-3-sulfate (see Fig. 55-8) and dehydroepiandrosterone sulfate (see Fig. 54-6)—breakdown products of sex steroids. Other anions, such as image and image, are excreted by anion exchangers.

N46-5

MRP2 (ABCC2)

Contributed by Emile Boulpaep, Walter Boron

A member of the ABC family (see Table 5-6), MRP2 has a broad substrate specificity but has the highest affinities for the bilirubin conjugated to monoglucuronide or diglucuronide. The affinity is also high for leukotriene C4.

The congenital deficiency of MRP2 causes Dubin-Johnson syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia (i.e., high levels of conjugated bilirubin in the blood).

References

Dubin IN, Johnson FB. Chronic idiopathic jaundice with unidentified pigment in liver cells: A new clinicopathological entity with a report of 12 cases. Medicine (Baltimore). 1954;33:155–197.

Nies AT, Keppler D. The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch. 2006;453:643–659.

Organic Cations

Biliary excretion of organic cations is poorly understood. MDR1 (ABCB1; see Table 5-6) is present in the canalicular membrane, where it secretes into the bile canaliculus (see Fig. 46-7) bulky organic cations, including xenobiotics, cytotoxins, anticancer drugs, and other drugs (e.g., colchicine, quinidine, verapamil, cyclosporine).

Other organic cations move into the canaliculus via the multidrug and toxin extrusion 1 (MATE1) transporters, which are driven by a pH gradient (see Fig. 46-7). The MATEs are one of the most highly conserved transporter families in nature, and MATE1 is highly expressed in many tissues. Thus, transcellular cation movement in liver is mediated by the combined action of electrogenic OCT-type uptake systems and MATE-type efflux systems.

In some cases, organic cations appear to move passively across the apical membrane into the canaliculus, sequestered by biliary micelles.

Biliary Lipids

Phospholipid is a major component of bile. MDR3 (ABCB4; see Table 5-6) is a flippase that promotes the active translocation of phosphatidylcholine (PC) from the inner to the outer leaflet of the canalicular membrane. Bile salts then extract the PC from the outer leaflet so that the PC becomes a component of bile, where it participates in micelle formation. Indeed, in humans with an inherited deficiency of MDR3, progressive liver disease develops, characterized by extremely low concentrations of phospholipids in the bile.

Lipid asymmetry in the canalicular membrane is essential for protection against the detergent properties of bile salts. The P-type ATPase ATP8B1 in the canalicular membrane translocates aminophospholipids—such as phosphatidylserine (PS) and phosphatidylethanolamine (PE)—from the outer to the inner leaflet of the bilayer, thereby leaving behind an outer leaflet that is depleted of PC, PS, and PE but enriched in sphingomyelin and cholesterol. The resulting lipid asymmetry renders the membrane virtually detergent insoluble and helps to maintain the functional complement of enzymes and transporters within the lipid bilayer. ATP8B1 is also present in the apical membranes of several other epithelia, including cholangiocytes and the epithelia of gallbladder, pancreas, and intestine. Mutations in ATP8B1 produce a chronic, progressive cholestatic liver disease (progressive familial intrahepatic cholestasis type 1).

Bile is also the main pathway for elimination of cholesterol. A heterodimer composed of the “half” ABC transporters ABCG5 and ABCG8 (see Table 5-6) is located on the canalicular membrane. This transporter is responsible for the secretion of cholesterol into bile. Although the mechanism is uncertain, the ABCG5/ABCG8 complex may form a channel for cholesterol translocation or alternatively may undergo a conformational change following ATP hydrolysis, thereby flipping a cholesterol molecule into the outer membrane leaflet in a configuration favoring release into the canalicular lumen. Mutations in the genes encoding either of the two ABC monomers lead to sitosterolemia, a disorder associated with defective secretion of dietary sterols into the bile, increased intestinal absorption of plant and dietary sterols, hypercholesterolemia, and early-onset atherosclerosis.

Hepatocytes take up proteins across their basolateral membranes by receptor-mediated endocytosis and fluid-phase endocytosis

The hepatocyte takes up macromolecules, such as plasma proteins, from the blood plasma through endocytosis, transports these molecules across the cytoplasm, and then secretes them into the bile through exocytosis. Three forms of endocytosis have been identified in the basolateral (sinusoidal) membrane: fluid-phase endocytosis (nonspecific), adsorptive endocytosis (nonspecific), and receptor-mediated endocytosis (specific). imageN46-6

N46-6

Protein Transport by Hepatocytes

Contributed by Fred Suchy

As noted in the text, hepatocytes take up proteins across their basolateral (i.e., sinusoidal) membranes via three forms of endocytosis. We discuss the fate of these endocytosed proteins in the next two paragraphs.

Intracellular Transport

Once proteins move into the hepatocyte by basolateral endocytosis, they can be transported across the cytoplasm within vesicles. This process, known as transcytosis, requires microtubules and is blocked by microtubule inhibitors, such as colchicine. Vesicular carriers transport the endocytosed proteins from the basolateral to the apical (i.e., canalicular) plasma membrane, where they exit via exocytosis. These same transcytotic vesicular carriers also ferry newly synthesized apical-membrane and secretory proteins. In the liver, most proteins destined for the apical membrane are initially transported from the trans-Golgi network to the basolateral membrane and subsequently transcytosed to the apical surface. It is believed that certain signal sequences on the protein (see p. 28) designate it as an apical membrane protein and are responsible for its correct targeting. The constitutive expression, rapid transport, and slow turnover of apical proteins mean that few are in the biosynthetic pipeline at steady state. During perturbations of liver function, such as cholestasis, the process of protein sorting is disturbed, and newly synthesized apical membrane proteins may accumulate in a subapical vesicular compartment or may be missorted to the basolateral domain.

Apical Exocytosis

The principal pathway for secretion of high-molecular-weight proteins, whether they originate de novo from within the hepatocyte or come from the plasma, is exocytosis at the apical membrane. Exocytosis may also be used to recruit transport proteins to the plasma membrane. For example, cAMP stimulates sorting of MRP2 (multidrug resistance–associated protein 2, ABCC2) to the canalicular membrane, a process that is accompanied by increases in canalicular membrane area and that can be inhibited by nocodazole, a microtubule inhibitor. Thus, the targeting of vesicles to the apical membrane may be a highly regulated (rather than constitutive) process and may be an important determinant of organic anion excretion into bile. A similar mechanism of both exocytosis and endocytosis at the apical membrane may be involved in the cell volume-regulatory responses to hyperosmotic stress (see p. 131) and hypo-osmotic stress (see pp. 131–132).

Fluid-phase endocytosis involves the uptake of a small amount of extracellular fluid with its solutes and is a result of the constitutive process of membrane invagination and internalization (see pp. 41–42). The process is nondiscriminatory and inefficient.

Adsorptive endocytosis involves nonspecific binding of the protein to the plasma membrane before endocytosis, and it results in more efficient protein uptake.

Receptor-mediated endocytosis is quantitatively the most important mechanism for the uptake of macromolecules (see p. 42). After endocytosis, the receptor recycles to the plasma membrane, and the ligand may be excreted directly into bile by exocytosis or delivered to lysosomes for degradation. Receptor-mediated endocytosis is involved in the hepatic removal from the blood of proteins such as insulin, polymeric immunoglobulin A (IgA), asialoglycoproteins, and epidermal growth factor.

 

SMALL INTESTINE

LARGE INTESTINE

Length (m)

6

2.4

Area of apical plasma membrane (m2)

~200

~25

Folds

Yes

Yes

Villi

Yes

No

Crypts or glands

Yes

Yes

Microvilli

Yes

Yes

Nutrient absorption

Yes

No

Active Na+ absorption

Yes

Yes

Active K+ secretion

No

Yes