Medical Physiology, 3rd Edition

Bile Formation

The secretion of canalicular bile is active and isotonic

The formation of bile occurs in three discrete steps. First, the hepatocytes actively secrete bile into the bile canaliculi. Second, intrahepatic and extrahepatic bile ducts not only transport this bile but also secrete into it a watery, image-rich fluid. These first two steps may produce ~900 mL/day of so-called hepatic bile (Table 46-2). Third, between meals, approximately half the hepatic bile—perhaps 450 mL/day—is diverted to the gallbladder, which stores the bile and isosmotically removes salts and water. The result is that the gallbladder concentrates the key remaining solutes in bile fluid—bile salts, bilirubin, cholesterol, and lecithin—by 10- to 20-fold. The 500 mL/day of bile that reaches the duodenum through the ampulla of Vater is thus a mixture of relatively “dilute” hepatic bile and “concentrated” gallbladder bile.

TABLE 46-2

Composition of Bile

PARAMETER

HEPATIC BILE

GALLBLADDER BILE

pH

7.5

6.0

Na+ (mM)

141–165

220

K+ (mM)

2.7–6.7

14

Ca2+ (mM)

1.2–3.2

15

Cl (mM)

77–117

31

image (mM)

12–55

19

Total phosphorus (g/L)

0.15

1.4

Bile acids (g/L)

3–45

32

Total fatty acids (g/L)

2.7

24

Bilirubin (g/L)

1–2

3

Phospholipids (g/L)

1.4–8.1

34

Cholesterol (g/L)

1–3.2

6.3

Proteins (g/L)

2–20

4.5

Data from Boyer JL: Mechanisms of bile secretion and hepatic transport. In Andreoli TE, Hoffman JF, Fanestil DD, Schultz SG (eds): Physiology of Membrane Disorders. New York, Plenum, 1986.

The first step in bile formation cannot be ultrafiltration because the hydrostatic pressure in the canaliculi is significantly higher than the sinusoidal perfusion pressure. This situation is in marked contrast to glomerular filtration by the kidney (see pp. 743–745), which relies predominantly on passive hydrostatic forces for producing the fluid in Bowman's space. Instead, bile formation is an active process. It is sensitive to changes in temperature and to metabolic inhibitors. Bile formation by hepatocytes requires the active, energy-dependent secretion of inorganic and organic solutes into the canalicular lumen, followed by the passive movement of water.

Canalicular bile is an isosmotic fluid. Water movement into the bile canaliculus can follow both paracellular and transcellular pathways. As far as the paracellular pathway is concerned, the movement of water through the tight junctions between hepatocytes carries with it solutes by solvent drag (see p. 467). Further down the biliary tree (i.e., ducts and gallbladder), where the pore size of paracellular junctions is significantly smaller, solvent drag is not as important. Organic solutes do not readily enter bile distal to the canaliculi.

As far as the transcellular pathway is concerned, water enters hepatocytes via aquaporin 9 (AQP9), found exclusively on the sinusoidal membrane. AQP9 also allows the passage of a wide variety of neutral solutes such as urea, glycerol, purines, and pyrimidines. The canalicular membrane expresses AQP8. Under basal conditions, AQP8 is predominantly localized to intracellular vesicles so that water permeability in the canalicular membrane is lower than that in the sinusoidal membrane and is rate limiting for transcellular water transport. However, upon cAMP stimulation, AQP8 from the intracellular pool inserts into the canalicular membrane, which substantially increased the water permeability of this membrane. With cAMP stimulation, transcellular water permeability in the hepatocyte is similar to that in the renal proximal tubule, where water flow is largely transcellular. Indeed, the transcellular pathway accounts for most of the water entering the bile canaliculus during choleresis.

Major organic molecules in bile include bile acids, cholesterol, and phospholipids

Bile has two important functions: (1) bile provides the sole excretory route for many solutes that are not excreted by the kidney, and (2) secreted bile salts and acids are required for normal lipid digestion (see pp. 925–929) and absorption (see pp. 929–933).

Both hepatic bile and gallbladder bile are complex fluids that are isosmotic with plasma (~300 mOsm) and consist of water, inorganic electrolytes, and a variety of organic solutes, including bilirubin, cholesterol, fatty acids, and phospholipid (see Table 46-2). The predominant cation in bile is Na+, and the major inorganic anions are Cl and image. Solutes whose presence in bile is functionally important include micelle-forming bile acids, phospholipids, and IgA.

Bile acids promote dietary lipid absorption through their micelle-forming properties (see p. 929). As shown in Figure 46-9, hepatocytes synthesize the so-called primary bile acids—cholic acid and chenodeoxycholic acid—from cholesterol. Indeed, biliary excretion of cholesterol and conversion of cholesterol to bile acids are the principal routes of cholesterol excretion and catabolism, so that bile formation is pivotal for total-body cholesterol balance. The first step in this conversion is catalyzed by cholesterol 7α-hydroxylase (CYP7A1),imageN46-7 a specific cytochrome P-450 enzyme located in the SER. As we see below, secondary bile acids are the products of bacterial dehydroxylation in the terminal ileum and colon. After being absorbed and returning to the liver (enterohepatic circulation, discussed below), these secondary bile acids may also undergo conjugation. Figure 46-9 shows typical examples of conjugation reactions.

image

FIGURE 46-9 Synthesis of bile acids. The liver converts cholesterol to the primary bile acids—cholic acid and chenodeoxycholic acid—in a series of 14 reactions occurring in four different cellular organelles. Bacteria in the terminal ileum and colon may dehydroxylate bile acids, yielding the secondary bile acids deoxycholic acid and lithocholic acid. The hepatocytes conjugate most of the primary bile acids to small molecules such as glycine and taurine before secreting them into the bile. In addition, those secondary bile acids that return to the liver via the enterohepatic circulation may also be conjugated to glycine or taurine

N46-7

Cholesterol 7α-Hydroxylase

Contributed by Fred Suchy

As noted in the text, the first step in the conversion of cholesterol to bile acids is the hydroxylation of cholesterol at position 7 by cholesterol 7α-hydroxylase. Bile acid levels regulate the activity of this enzyme, probably by both positive and negative feedback. Negative feedback in a cultured rat hepatocyte model occurs partly at the transcriptional level.

Phospholipids in bile help to solubilize cholesterol as well as diminish the cytotoxic effects of other bile acids on hepatocytes and bile duct cells. IgA inhibits bacterial growth in bile.

Excretory or waste products found in bile include cholesterol, bile pigments, trace minerals, plant sterols, lipophilic drugs and metabolites, antigen-antibody complexes, and oxidized glutathione.

Bile is also the excretory route for compounds that do not readily enter the renal glomerular filtrate, either because they are associated with proteins such as albumin or because they are associated with formed elements in blood. Although these compounds are generally lipophilic, they also include the heavy metals. Some bile acids (e.g., the trihydroxy bile acid cholic acid) are only partly bound to serum albumin and may therefore enter the glomerular filtrate. However, they are actively reabsorbed by the renal tubule. In health, bile acids are virtually absent from the urine.

Canalicular bile flow has a constant component driven by the secretion of small organic molecules and a variable component driven by the secretion of bile acids

Total bile flow is the sum of the bile flow from hepatocytes into the canaliculi (canalicular flow) and the additional flow from cholangiocytes into the bile ducts (ductular flow). In most species, the rate of canalicular bile secretion (i.e., milliliters per minute) increases more or less linearly with the rate of bile acid secretion (i.e., moles per minute). Canalicular bile flow is the sum of two components (Fig. 46-10): (1) a “constant” component that is independent of bile acid secretion (bile acid–independent flow) and (2) a rising component that increases linearly with bile acid secretion (bile acid–dependent flow). In humans, most of the canalicular bile flow is bile acid dependent. If we now add the ductular secretion, which is also “constant,” we have the total bile flow in Figure 46-10. We discuss the canalicular secretion in the remainder of this section and ductular secretion in the following section.

image

FIGURE 46-10 Components of bile flow.

Bile Acid–Independent Flow in the Canaliculi

The secretion of organic compounds probably provides the major driving force for bile acid–independent flow. For example, glutathione, present in bile in high concentrations, may generate a potent osmotic driving force for canalicular bile formation. imageN46-8

N46-8

Contribution of Inorganic Solutes to Bile Acid–Independent Flow

Contributed by Fred Suchy

In addition to organic solutes, inorganic solutes also contribute to bile acid–independent flow of bile. The secretion of these inorganic electrolytes occurs primarily by solvent drag (see p. 467) and passive diffusion (e.g., through canalicular Cl channels; see Fig. 46-5B). To the extent that these inorganic electrolytes enter the canalicular lumen by passive diffusion, they pull in water osmotically and thus contribute to bile acid–independent flow. However, this is not a major effect.

Bile Acid–Dependent Flow in the Canaliculi

The negatively charged bile salts in bile are in a micellar form and are—in a sense—large polyanions. Thus, they are effectively out of solution and have a low osmotic activity coefficient. However, the positively charged counterions accompanying these micellar bile acids are still in aqueous solution and may thus represent the predominant osmotic driving force for water movement in bile acid–dependent flow. If one infuses an animal with a nonphysiological bile acid that does not form micelles or one that forms micelles only at a rather high concentration, the osmotic activity will be higher, and thus the exogenous bile acid will be more effective in producing bile acid–dependent flow. In other words, the slope of the blue bile acid–dependent line in Figure 46-10 would be steeper than for physiological bile acids.

Bile flow does not always correlate with the osmotic activity of the bile acid. In some cases, bile acids increase electrolyte and water flux by other mechanisms, such as by stimulating Na+-coupled cotransport mechanisms or by modulating the activity of other solute transporters. For example, the bile acid ursodeoxycholic acid produces a substantial increase in bile flow by markedly stimulating biliary image excretion.

Bile acids in the lumen may also stimulate the secretion of other solutes by trapping them in the lumen. These solutes include bilirubin and other organic anions, as well as lipids such as cholesterol and phospholipids. The mixed micelles formed by the bile acids apparently sequester these other solutes, thus lowering their effective luminal concentration and favoring their entry. Therefore, excretion of cholesterol and phospholipid is negligible when bile acid output is low, but it increases and approaches maximum values as bile acid output increases.

Secretin stimulates the cholangiocytes of ductules and ducts to secrete a watery, image-rich fluid

As discussed in the previous section, biliary epithelial cells, or cholangiocytes, are the second major source of the fluid in hepatic bile. Experimentally, one can isolate cholangiocytes from normal liver or from the liver of experimental animals in which ductular hyperplasia has been induced by ligating the bile duct. These cholangiocytes (Fig. 46-11) have 6 of the 13 known human aquaporins, an apical Cl-HCO3exchanger AE2, and several apical Cl channels, including the cystic fibrosis transmembrane conductance regulator (CFTR; see p. 120). In a mechanism that may be similar to that in pancreatic duct cells (see Fig. 43-6), the Cl-HCO3 exchanger, in parallel with the Cl channels for Cl recycling, can secrete an image-rich fluid. imageN46-9 AQP1, CFTR, and AE2 colocalize to intracellular vesicles in cholangiocytes; secretory agonists cause all three to co-redistribute to the apical membrane.

image

FIGURE 46-11 Secretion of an image-rich fluid by cholangiocytes. Secretin, glucagon, VIP, and gastrin-releasing peptide (GRP) all are choleretics. Somatostatin either enhances fluid absorption or inhibits secretion. CA, carbonic anhydrase.

N46-9

image Secretion by Cholangiocytes

Contributed by Fred Suchy

As noted in the text, the mechanism of image secretion by cholangiocytes may be similar to that of secretion by pancreatic duct cells. For a discussion of the latter process, see imageN46-20.

In addition, cholangiocytes transport water into bile via an aquaporin water channel. Na-H exchangers are expressed on both the basolateral membrane (NHE1) and the apical membrane (NHE2) of cholangiocytes.

N46-20

image Secretion by the Pancreatic Duct

Contributed by Emile Boulpaep, Walter Boron

The current model for image secretion by the pancreatic duct is very similar to that outlined in Figure 43-6. However, we can now add some important details about the apical step of image secretion. The Cl-HCO3 exchanger at the apical membrane is a member of the SLC26 family (Mount and Romero, 2004)—previously known as the SAT family—specifically, SLC26A6 (also known as CFEX). We now appreciate that SLC26A6, which is capable of exchanging several different anions (e.g., Climage, oxalate), is electrogenic (Jiang etal, 2002). When mediating Cl-HCO3 exchange, it appears that SLC26A6 exchanges two image ions for every Cl ion. This stoichiometry would strongly favor the efflux of image across the apical membrane of the pancreatic duct cell.

As noted in the text, the Cl that enters the cell via SLC26A exits the cell via apical Cl channels, principally CFTR. Interestingly, it appears that an interaction between the SLC26A6 protein and CFTR greatly increases the open probability of CFTR (Ko etal, 2004).

Another member of the SLC26 family—SLC26A3—also is present in the apical membrane of pancreatic duct cells. SLC26A3 is also electrogenic but has a stoichiometry opposite to that of SLCA6, two Clions for every image. This transporter would extrude Cl (and take up image) from the duct cell across the apical membrane. Its physiological function might be to reabsorb image at times when the duct is not secreting image or to contribute to the recycling of Cl when the duct is secreting image.

References

Jiang Z, Grichtchenko II, Boron WF, Aronson PS. Specificity of anion exchange mediated by mouse Slc26a6. J Biol Chem. 2002;277:33963–33967.

Ko SB, Zeng W, Dorwart MR, et al. Gating of CFTR by the STAS domain of SLC26 transporters. Nat Cell Biol. 2004;6:343–350.

Mount DB, Romero MF. The SLC26 gene family of multifunctional anion exchangers. Pflugers Arch. 2004;447:710–721.

A complex network of hormones, mainly acting via cAMP, regulates cholangiocyte secretory function. Secretin receptors (see pp. 886–887) are present on the cholangiocyte basolateral membrane, a fact that explains why secretin produces a watery choleresis—that is, a bile rich in image (i.e., alkaline) but poor in bile acids. The hormones glucagon (see pp. 1050–1053) and vasoactive intestinal peptide (VIP; see Fig. 13-9) have similar actions. imageN46-10 These hormones raise [cAMP]i and thus stimulate apical Cl channels and the Cl-HCO3 exchanger. A Ca2+-activated Cl channel is also present in the apical membrane. imageN46-11

N46-10

Regulation of Cholangiocyte Secretion

Contributed by Fred Suchy

In addition to the hormones mentioned on pages 960–961gastrin-releasing peptide (GRP) also stimulates fluid and image secretion from cholangiocytes, but through mechanisms other than cAMP, cGMP, and Ca2+. Exposure of polarized cholangiocytes to ATP results in luminal secretion through activation of P2µ receptors on the apical membrane. Release of ATP into bile appears to serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.

N46-11

Ca2+-Activated Cl Channels

Contributed by Emile Boulpaep, Walter Boron

Ca2+-activated Cl channels (CaCCs) play important physiological roles. One group of CaCCs are encoded by the ANO genes (see Table 6-2, family No. 17). The bestrophins, encoded by at least four BEST genes, constitute another family of CaCCs. The molecular identity of CaCCs in the liver is unknown.

References

Hartzell HC, Putzier I, Arreola J. Calcium-activated chloride channels. Annu Rev Physiol. 2005;67:719–758.

Hartzell HC, Qu Z, Yu K, et al. Molecular physiology of bestrophins: Multifunctional membrane proteins linked to Best disease and other retinopathies. Physiol Rev. 2008;88:639–672.

Koumi S, Sato R, Aramaki T. Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes. J Gen Physiol. 1994;104:357–373.

Cholangiocytes are also capable of reabsorbing fluid and electrolytes, as suggested by the adaptation that occurs after removal of the gallbladder (i.e., cholecystectomy). Bile found within the common bile duct of fasting cholecystectomized animals is similar in composition to the concentrated bile typically found in the gallbladder. Thus, the ducts have partially taken over the function of the gallbladder (see below).

The hormone somatostatin inhibits bile flow by lowering [cAMP]i, an effect opposite that of secretin. This inhibition may be caused by enhancing fluid reabsorption by bile ducts or by inhibiting ductular secretion of the image-rich fluid discussed above.

Solutes reabsorbed from bile by cholangiocytes are recycled. As shown in Figure 46-2, the intralobular bile ducts are endowed with a rich peribiliary vascular plexus that is supplied by the hepatic artery. The blood draining this plexus finds its way into the hepatic sinusoids. This plexus is analogous to the capillaries of the gut, which, via the portal vein, also find their way into the hepatic sinusoids. Thus, some solutes, such as the hydrophilic bile acid ursodeoxycholic acid, may be absorbed by the cholangiocytes from bile and returned to the hepatocytes for repeat secretion, a process that induces significant choleresis.

The gallbladder stores bile and delivers it to the duodenum during a meal

The gallbladder is not an essential structure of bile secretion. Tonic contraction of the sphincter of Oddi facilitates gallbladder filling by maintaining a positive pressure within the common bile duct. As we noted above, up to 50% of hepatic bile—or ~450 mL/day—is diverted to the gallbladder during fasting. The remaining ~450 mL/day passes directly into the duodenum. Periods of gallbladder filling between meals are interrupted by brief periods of partial emptying of concentrated bile and probably aspiration of dilute hepatic bile in a process analogous to the function of a bellows.

Gallbladder emptying and filling is under feedback control. During feeding, CCK secreted by duodenal I cells (see Table 41-1) causes gallbladder contraction and the release of bile into the duodenum, where the bile promotes fat digestion and suppresses further CCK secretion. On reaching the ileum, bile acids induce synthesis of fibroblast growth factor 19 (FGF19); FGF19, after transit in portal blood, causes relaxation of gallbladder smooth muscle, which allows gallbladder refilling. Thus, CCK and FGF19 control the periodicity of gallbladder emptying and filling.

During the interdigestive period, the gallbladder concentrates bile acids—and certain other components of bile—up to 10- or even 20-fold within the gallbladder lumen because they are left behind during the isotonic reabsorption of NaCl and NaHCO3 by the leaky gallbladder epithelium (Fig. 46-12). The apical step of NaCl uptake and transport is electroneutral and is mediated by parallel Na-H and Cl-HCO3exchangers. At the basolateral membrane, Na+ exits through the Na-K pump, whereas Cl most likely exits by Cl channels. Both water and image move passively from lumen to blood through the tight junctions. Water can also move through the cell via AQP1 (expressed on apical and basolateral membranes) and AQP8 (found only apically). The net transport is isotonic, which leaves behind gallbladder bile that is also isotonic but has a higher concentration of bile salts, K+, and Ca2+. Net fluid and electrolyte transport across the gallbladder epithelium is under hormonal regulation. Both VIP (released from neurons innervating the gallbladder) and serotonin inhibit net fluid and electrolyte absorption. Conversely, α-adrenergic blockade of neuronal VIP release increases fluid absorption.

image

FIGURE 46-12 Isotonic fluid reabsorption by the gallbladder epithelium.

Although the gallbladder reabsorbs NaCl by parallel Na-H and Cl-HCO3 exchange at the apical membrane, Na-H exchange outstrips Cl-HCO3 exchange; the end result is net secretion of H+ ions. This action neutralizes the image and acidifies the bile. The H+ secreted by the gallbladder protonates the intraluminal contents. This action greatly increases the solubility of calcium salts in bile and reduces the likelihood of calcium salt precipitation and gallstone formation. Common “pigment gallstones” contain one or more of several calcium salts, including carbonate, bilirubinate, phosphate, and fatty acids. The solubility of each of these compounds is significantly increased by the acidification of bile.

Mucus secretion by gallbladder epithelial cells results in the formation of a polymeric gel that protects the apical surface of the gallbladder epithelium from the potentially toxic effects of bile salts. However, excessive mucin synthesis can be deleterious. For example, in animal models of cholesterol cholelithiasis (i.e., formation of gallstones made of cholesterol), a marked increase in mucin release precedes crystal and stone formation.

The relative tones of the gallbladder and sphincter of Oddi determine whether bile flows from the common hepatic duct into the gallbladder or into the duodenum

Bile exiting the liver and flowing down the common hepatic duct reaches a bifurcation that permits flow either into the cystic duct and then into the gallbladder or into the common bile duct, through the sphincter of Oddi, and into the duodenum (see Fig. 46-4). The extent to which bile takes either path depends on the relative resistances of the two pathways.

The sphincter of Oddi—which also controls the flow of pancreatic secretions into the duodenum—corresponds functionally to a short (4- to 6-mm) zone within the wall of the duodenum. The basal pressure within the lumen of the duct at the level of the sphincter is 5 to 10 mm Hg. The pressure in the lumen of the resting common bile duct is also 5 to 10 mm Hg, compared with a pressure of ~0 mm Hg inside the duodenum.

The basal contraction of the sphincter prevents reflux of the duodenal contents into the common bile duct. In its basal state, the sphincter exhibits high-pressure, phasic contractions several times per minute. These contractions are primarily peristaltic and directed in antegrade fashion to provide a motive force toward the duodenum. Thus, the sphincter of Oddi acts principally as an adjustable occluding mechanism and a regulator of bile flow.

Both hormonal and cholinergic mechanisms appear to be involved in gallbladder emptying. Dietary lipid stimulates the release of CCK from duodenal I cells (see pp. 889–890). This CCK not only stimulates pancreatic secretion but also causes smooth-muscle contraction and evacuation of the gallbladder. The coordinated response to CCK also includes relaxation of the sphincter of Oddi, which enhances bile flow into the duodenum (Box 46-2).

Box 46-2

Cholestasis

The term cholestasis refers to the suppression of bile secretion. Biliary constituents may therefore be retained within the hepatocyte and regurgitated into the systemic circulation. Cholestasis causes three major groups of negative effects: first, regurgitation of bile components (bile acids, bilirubin) into the systemic circulation gives rise to the symptoms of jaundice and pruritus (itching). Second, cholestasis damages hepatocytes, as evidenced by the release of clinically important liver enzymes (e.g., alkaline phosphatase) into the plasma. Third, because the bile acids do not arrive in the duodenum, lipid digestion and absorption may be impaired.

Many acute and chronic liver diseases produce cholestasis by mechanically obstructing the extrahepatic bile ducts or by impairing bile flow at the level of the hepatocytes or intrahepatic bile ducts. The mechanisms underlying the obstructive and functional forms of cholestasis are complex and have not been completely defined. Experimental modeling of cholestasis has produced multiple abnormalities: (1) altered plasma-membrane composition and fluidity; (2) inhibition of membrane proteins, including the Na-K pump and aquaporins; (3) reduced expression of genes encoding transporters for bile acids and other organic anions; (4) altered expression of nuclear receptors and associated epigenetic modifications that regulate transporters; (5) increased permeability of the paracellular pathway, with backdiffusion of biliary solutes into the plasma; (6) altered function of microfilaments, with decreased contractions of bile canaliculi; and (7) loss of the polarized distribution of some plasma-membrane proteins. Cholestatic conditions, such as bile duct obstruction, markedly increase the basolateral expression of MRP4 and MRP6 as well as OSTα-OSTβ—which normally are expressed only minimally. The induction of these transporters allows the efflux of bile acids and other cholephilic anions from the hepatocyte into sinusoidal blood.

 

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