There also is segmental heterogeneity along the crypt-villus axis. Stem cells near the base of the crypt differentiate and migrate upward to form villus enterocytes in the small intestine or surface colonocytes in the large intestine while undergoing important changes in their transport and barrier properties (Fig. 99-3).¹¹^,¹² As epithelial cells migrate away from the proliferative zone, the complexity of their tight junctions increases, the microvillus architecture of their apical membrane becomes more pronounced, and underlying cytoskeleton and signaling molecules undergo change; there also is increased expression of Na⁺ nutrient-coupled transporters, apical Na⁺-H⁺ exchangers, and brush border membrane hydrolases. In contrast, the levels of the Na⁺ pumps remain relatively constant and others, such as the signaling molecule adenylate cyclase and the cyclic adenosine monophosphate (cAMP)-associated Cl⁻ channel CFTR (cystic fibrosis transmembrane conductance regulator), decrease in more mature villus cells.

Figure 99-3. Types of epithelial cells of the intestinal mucosa: enterocytes, endocrine cells, goblet cells, and Paneth cells. All of these cell types originate from the proliferative zone near the base of the intestinal crypt. With the exception of Paneth cells, these cells migrate up the villus axis, mature during this process, and eventually undergo apoptosis and slough after three to five days at the tip of the villus. Paneth cells remain at the base of the crypt and make defensins, which are important in host defense.

This spatial distribution of transporters (Fig. 99-4) is consistent with a model in which secretory function resides primarily in the crypts and absorption occurs in villus or surface cells. This dichotomy between absorptive surface cells and secretory crypt cells, however, is far from absolute; for example, in the colon, crypts absorb Na⁺ and fluid, and surface cells secrete Cl⁻.¹³^,¹⁴ Thus, depending on their relative position along the crypt-villus axis, the crosstalk between transporters and their signaling molecules can vary and fine tune intestinal function. This segregation of absorptive and secretory functions might explain why, in diseases that selectively damage villi or surface epithelia—such as enteric infection, inflammatory bowel disease (IBD), and celiac disease—secretion predominates.

Figure 99-4. Spatial location of transport protein gradients. There is a significant spatial geometry of transport proteins along the crypt-villus (crypt-surface) axis. Some transport proteins are found at relatively constant concentrations along this axis, whereas some proteins exhibit a greater density in the base of the crypt and others are denser toward the villus or surface. CFTR, cystic fibrosis transmembrane conductance regulator; DRA, down-regulated in adenoma; NHE, sodium-hydrogen exchanger; PAT, putative anion transporter.

MOVEMENT ACROSS THE INTESTINAL EPITHELIUM

Movement of ions and solutes across the epithelium is bidirectional and occurs via the transcellular and paracellular routes. Paracellular movement is largely passive, in response to a variety of gradients, including concentration, electrical, osmotic, and hydrostatic; transcellular movement of ions and solutes occurs by active and passive transport mechanisms. Net transport is termed absorptive if the mucosal-to-serosal flux (J_ms) is greater than the serosal-to-mucosal (J_sm) flux and, and it is termed secretory if J_sm > J_ms. Changes in either or both can alter the direction of the net movement; for example, the ileum, which normally exhibits an absorptive flux, responds to cholera toxin with a decrease in J_ms and an increase in J_sm for Cl⁻, resulting in massive fluid secretion.

Characteristics of the tight junctions—for example, tight versus leaky—vary along the length of the intestine and dictate the contribution of paracellular fluxes to overall transport. The effectiveness of a transepithelial gradient may be modified by series of physical barriers, including an unstirred layer created by the glycocalyx above the apical membrane, the lipid composition of the apical and basolateral membrane, the tight junctions, the geometry of the basolateral space between cells, and the basement membrane. Generally, movement of an uncharged particle is dictated solely by concentration gradients. In contrast, the transport of an ion is governed by the electrical potential and concentration differences (the electrochemical gradient) across the transported surface. Solvent drag, a nonspecific entraining of solutes along with the movement of water across paracellular pathways, is an absorptive mechanism that may be especially important in the small intestine, for example, for Na⁺-coupled solute absorption.

TIGHT AND LEAKY EPITHELIA

The paracellular space and junctional complexes between cells define the barrier function of epithelia. Epithelia with a low transepithelial voltage and low resistance are considered leaky, and those that exhibit a high transepithelial voltage and high resistance are considered tight. The tight junctions in villi have higher resistance than do those in crypts. Transepithelial resistance increases in a cephalocaudal direction (see Fig. 99-1).¹³

Since the 1990s, the model of paracellular transport and tight junctions has rapidly evolved from a static rigid barrier to a dynamic complex structure that is finely regulated (see Fig. 99-2). Movement through the space is exclusively passive, but it is influenced by electrical conductivity, charge selectivity, and its ability to be regulated. Cell-to-cell communications along the paracellular pathway occur in several discrete structures: zona occludens (ZO; tight junction), zona adherens (ZA), desmosomes, and gap junctions. The ZO is composed of several families of proteins that determine its physical and biological properties. For example, claudins belong to a family of 24 membrane-spanning proteins (24-27 kd) that form pores by interactions of the extracellular domains of claudins of adjoining cells; homotypic adhesion claudins are important in determining the charge selectivity of the tight junction.¹⁵^,¹⁶

Additional proteins in the tight junction include occludins, junctional adhesion molecules (JAMs), and scaffolding proteins such as the zona occludens proteins (ZO-1, ZO-2) and multi-PDZ domain protein 1 (MUPP-1). The scaffolding proteins serve to link membrane proteins to an array of protein kinases, phosphatases and, via filamentous actin, to myosin in the terminal web, thereby influencing paracellular permeability.¹⁷^,¹⁸ For example, disruption of tight junctions by enteropathogenic Escherichia coli is specifically associated with protein kinase Cζ activation.¹⁹ Another junctional complex that allows cell-to-cell interaction is the zona adherens. In epithelia, the zona adherens primarily is made up of E-cadherins, 120-kd transmembrane glycoproteins, with extracellular motifs that engage in calcium-dependent homotypic interaction with cadherins of adjoining cells. Intracellularly, cadherins bind to a family of adhesion molecules, the catenins, which in turn anchor to a dense actin-filament network. Alterations in cadherin-catenin distribution or function have been implicated in carcinogenesis.²⁰

Desmosomes are junctional complexes that are structurally similar to zona adherens junctions, although instead of actin, they link to intermediate filaments through a dense plaque of intracellular anchor proteins. Gap junctions have a unique function: They bridge gaps between cells, thus allowing neighboring cells to exchange small molecules. They are made up of an assembly of connexins, a four-pass membrane-spanning protein, six of which join to form a hemichannel. When these hemichannels in two adjoining cells are aligned, they form a continuous pore that connects the interior of the two cells.¹

TRANSEPITHELIAL TRANSPORT

Our current understanding of the movement of ions, solutes, and fluid across epithelia is gleaned from a combination of in vitro studies using reductionist models of cell lines or isolated epithelial sheets, and from complex in vivo methodologies such as the triple-lumen perfusion technique. All these models underscore that transepithelial ion (largely Na⁺) movement from the mucosa to the serosa drives fluid absorption, whereas net ion (largely Cl⁻) movement in the reverse direction drives fluid secretion. Although different approaches help elucidate a complex mechanism, at times they give confounding results. For example, some in vitro studies report decreased Cl⁻ secretion and increased Na⁺ absorption in the jejunum of cystic fibrosis patients, implying that the intestinal manifestations of the disease are due to hyperabsorption of water. In contrast, in vivo studies show decreases in both Cl⁻ secretion and passive Cl⁻ absorption, suggesting that rather than a hyperabsorption of fluid, the severity of the disease is reflected by decreased fluid absorption.²¹

The reductionist models allow us to focus on transport processes at the cellular and paracellular level. In the intact intestine, however, things are more complicated. The geometry of the intestinal wall and the unstirred layer influence the distance that an individual molecule must traverse to reach the apical membrane. The extracellular glycosylated domains of apical membrane proteins make up the glycocalyx, which contributes to the thickness and permeability of the unstirred layer; this layer can be a diffusive barrier to the movement of large lipophilic molecules in a chiefly aqueous milieu. Physical parameters such as the mixing of luminal contents by peristalsis, villus motility, and the finer movement of the microvilli influence this rate.

TRANSCELLULAR TRANSPORT

Transcellular transport of ions and solutes can be passive or active. Because of the semipermeable nature of the lipid membrane, movement through the cell requires the deployment of specialized membrane proteins, such as channels, carriers, and pumps. The negative intracellular potential favors cation entry into, and anion exit from, the cell. This leads to the curious situation in which ions can move passively against their concentration gradient. For example, although the chemical concentration of Cl⁻ in the cell is relatively low (~35 mmol) compared with the outside concentration (~110 mmol), the intracellular electronegativity creates a driving force for Cl⁻ exit out of the cell.

WATER MOVEMENT

Although water movement is a major property of the intestine, the mechanism(s) of intestinal water transport have not been clearly delineated. The movement of water is inextricably linked to the movement of solutes, in response to osmotic gradients. The standing-gradient hypothesis of water absorption suggests that even a small increase (2-3 mOsm) in the osmolarity of the intercellular and subepithelial spaces can cause movement of water across the epithelium, both through and around the cells.²² In the early 1950s, “water pores” were postulated to explain transepithelial water movement, but it was not until the remarkable discovery of the aquaporin (AQP) family of water transporters that a role of specific membrane proteins was implicated in erythrocyte and renal water transport.²³^,²⁴ Although AQP2, AQP3, and AQP7 have been localized to the small intestine and AQP1, AQP3, AQP4, and AQP8 have been localized to the large intestine, the specific intestinal apical water channel, if any, has eluded discovery.²⁵ Wright and associates proposed that the apical Na⁺-glucose transporter (SGLT) also may be able to transport water, perhaps as much as 5 L/day,²⁶^,²⁷ but whether this can compensate for the puzzling lack of functional apical AQPs remains to be determined.

CHANNELS, CARRIERS, AND PUMPS

Small hydrophobic and uncharged molecules move across the lipid bilayer of the cell by diffusion, the rate of transport determined by the concentration gradients and diffusion coefficients (Fig. 99-5). Oxygen, carbon dioxide, fat-soluble vitamins, and unconjugated bile acids are examples of substances transported by diffusion. Because the majority of ions and solutes cannot cross the phospholipid membrane by diffusion, the cell employs an array of distinct integral membrane proteins, including channels, carriers, and pumps to cross cell membranes (Fig. 99-5).¹

Figure 99-5. Channels, carriers, and pumps. Because only nonpolar solutes freely cross a lipid domain by simple diffusion (A), transfer of ions and charged molecules necessitates specific transmembrane proteins to modulate entry and exit. Ion-specific channels mediate membrane transport by facilitated diffusion (B). Carriers permit facilitated diffusion and transfer specific solutes across the membrane by undergoing a conformational change (C). Transcellular transport of water molecules occurs via channel proteins or carrier proteins (D). Active transport occurs against an electrochemical gradient and can be driven by adenosine triphosphate (ATP) (primary active transport; E1) or an ionic gradient (secondary active transport; E2)

Channels are pores that allow the swift (>10⁶ ions/sec) and controlled (by rapid opening and closing) transit of ions across the membrane, driven by the electrochemical gradient. The advent of molecular cloning techniques, patch clamp methodology (which allows the measurement of function of single channels), and membrane protein crystallography has greatly advanced our knowledge of how these proteins function. Channels tend to be ion selective. For example, Na⁺ channels exclude K⁺ despite its same charge and smaller size. Selectivity is determined by the hydration radius of the ion and the physiochemical nature of the pore. The overall transport of a particular ion is determined by the electrochemical gradient, the density of channels, and the gating (open-close time) of the channel; gating may be modulated by voltage, ion, concentration, or intracellular regulation. Mutations of critical residues in the channel protein can have dire functional consequences; for example, in cystic fibrosis, specific mutations of the CFTR affect the ability to transport chloride and bicarbonate.²⁸^,²⁹

Carriers are another class of integral membrane proteins responsible for transport of ions and solutes at rates several orders of magnitude lower than channels. Carrier-mediated transport exhibits substrate specificity, saturation, and inhibitory kinetics. Carriers undergo a series of sequential conformational changes to facilitate the transport of substrates across a membrane. When concentration or electrochemical gradients drive carrier-mediated transport, the process is downhill and is termed facilitated diffusion. For example, the entry of fructose into the enterocyte via Glut-5 is by facilitated diffusion. The entering fructose is rapidly isomerized to glucose, maintaining the downhill gradient for fructose.

In contrast, other carriers harness the electrochemical energy established by the downhill movement of a second ion, usually Na⁺, to move a solute or another ion uphill. This process is termed secondary active transport because the specific gradient is indirectly created by a distinct energy-using process. For example, glucose uptake via the apical membrane Na⁺-dependent glucose transporter, SGLT, is driven by the Na⁺ gradient generated by the basolateral Na⁺,K⁺-ATPase. Carriers exhibit substrate specificity; thus, SGLT transports d-glucose but not l-glucose. Equally important, carriers can transport single or multiple substrates and perform the transport in different directions. Uniporters, such as Glut-2 in the basolateral membrane, transport one type of substrate, hexoses. Symporters, such as the Na⁺/K⁺/2Cl⁻ cotransporter, move Na⁺, K⁺, and Cl⁻ in the same direction, whereas antiporters, such as the Na⁺-H⁺ exchangers, move the two ions in opposite directions.

Pumps are the third class of integral membrane proteins and directly use energy, generally adenosine triphosphate (ATP) hydrolysis, to move ions against an electrochemical gradient. This process is termed primary active transport. Na⁺,K⁺-ATPase is the quintessential pump in the intestine. Other pumps important in GI epithelial transport include the luminal gastric and colonic H⁺,K⁺-ATPases and the basolateral Ca²⁺-ATPases.

ION TRANSPORTERS

APICAL SODIUM CHANNEL

In the GI tract, the surface epithelial cells of the distal colon and rectum exhibit electrogenic Na⁺ absorption against a fairly steep concentration gradient. The downhill electrochemical gradient created by the Na⁺ pump drives Na⁺ entry via an apical membrane Na⁺-specific ion channel (Fig. 99-6), which belongs to the family of epithelial Na⁺ channels (ENaCs). Members of the ENaC family are found in many epithelia.³⁰^–³² They are multimeric proteins composed of α, β, and γ subunits; they exhibit a high sensitivity to the diuretic amiloride; and they are stimulated by mineralocorticoids and cAMP. Colonic ENaCs are inhibited by increases in intracellular Ca²⁺. Unlike many other channels that regulate transport by gating, ENaCs modulate transport by varying the channel density in the cell membrane; this variation may be accomplished through changes (increases or decreases) in synthesis (e.g., aldosterone) or exocytosis (e.g., cAMP, vasopressin) of the channels. Additionally, both aldosterone and cAMP block the association of ENaC with Nedd4-2, a ubiquitin protein ligase, which normally flags the protein for degradation; this block of degradation increases ENaC. Mutations in this pathway can result in the increased Na⁺ absorption and hypertension characteristic of Liddle’s syndrome, an autosomal dominant disorder with features of hyperaldosteronism that is a cause of infantile hypertension.

Figure 99-6. Apical sodium transporters. Sodium crosses the apical membrane of the epithelial cell down an electrochemical gradient. The mechanisms may be (1) an ion-specific channel that can be blocked by amiloride; (2) a carrier (e.g., SGLT1) that couples the movement of sodium and a nutrient such as glucose; or (3) a carrier (antiport carrier; e.g., NHE3) that allows electroneutral entry of sodium in exchange for intracellular hydrogen. The common exit pathway across the basolateral membrane is the Na⁺ pump (4). K⁺ channels help maintain the electrochemical gradient (5). Cl⁻ moves passively through the paracellular pathway or via cellular transporters (6). Glucose exits the basolateral membrane via the facilitated diffusion hexose transporter, GLUT2. NHE3, sodium-hydrogen exchanger 3; SGLT1, sodium-glucose cotransporter 1.

NUTRIENT-COUPLED SODIUM TRANSPORT

By and large, nutrient transporters are the purview of the small intestine. Transport of many hydrophilic nutrients including glucose, amino acids, and some vitamins occurs against their concentration gradients via secondary active transport at the apical membrane and facilitated diffusion across the basolateral membrane.

Glucose transport processes, elegantly elucidated by Wright and coworkers, provide a good example of nutrient transport.³³ Transport across SGLT1 is electrogenic (2 Na⁺ to 1 glucose), is stereospecific (d-isomer), and transports galactose but not fructose.³³ Glucose exit across the basolateral membrane occurs via a separate family of facilitated diffusion carriers, the glucose transporters (GLUT-2) (see Fig. 99-6). Fructose enters the cell via another member of this family, GLUT-5, and exits via GLUT-2.

Although it is clear that Na⁺ and glucose absorption stimulates water absorption, the mechanism is not fully delineated. The classic explanation is that basolateral exit of glucose creates a hypertonic compartment in the paracellular space, thereby generating an osmotic gradient for the entry of fluid from the lumen. Some enticing evidence suggests that secondary to transcellular transport through SGLT, passive processes triggers the contraction of the actomyosin ring in the terminal web, resulting in increased paracellular permeability to glucose and to water. Additionally, SGLT activation results in a protein kinase-dependent recruitment of GLUT2 to the apical membrane, which then serves as a high-capacity, low-affinity route for sugar entry during feeding.³⁴^,³⁵ Evidence suggests that SGLT can serve as a water channel (210 water to 2 Na⁺ to 1 glucose) and might account for up to 5 L of fluid reabsorption in the fed state.³⁶ How molecular regulation of transporters is translated into net nutrient absorption during feeding in vivo is a critical area of inquiry. For a description of similar advances made in our understanding of amino acid and vitamin transport, see Chapters 100 and 101.

SODIUM-HYDROGEN EXCHANGERS

The exchange of extracellular Na⁺ for intracellular H⁺ is a process that is driven by the electrochemical gradient for Na⁺ and by a pH gradient resulting from a moderately acidic intracellular environment; this process occurs in almost every cell. In mammalian intestine, members of the Na⁺-H⁺ exchange (NHE) gene family play an important role in electroneutral sodium absorption. Electroneutral Na⁺ absorption may be down-regulated during eating and increases upon postprandial nutrient absorption.

Ten mammalian isoforms of NHE have been cloned. NHE1-4 and 6-9 exhibit species- and segment-specific distribution in the GI tract.³⁷^–³⁹ NHE1 is a plasma membrane protein found in epithelial and nonepithelial cells. It is expressed on epithelial basolateral membranes and functions as the housekeeper regulator of intracellular pH, cell volume, and growth. NHE2 and NHE3 are apical membrane proteins restricted to epithelia and are the major conduits for electroneutral Na⁺ absorption in the intestine (see Fig. 99-6). NHE2 is expressed throughout the GI tract, but maximal expression is in the proximal colon. NHE3 is considered a marker for the absorptive cells of the small intestine and colon; it is expressed only in the villus or surface cells, and not in the crypts. NHE4 is located in the basolateral membrane and is primarily expressed in gastric parietal and chief cells, where it might have a role in acid secretion. NHE5 and NHE10 are not expressed in the GI tract, and the roles of NHE6-9 remain to be determined.

NHE activity is differentially modulated by neural, paracrine, or endocrine stimuli through intricate scaffolding complexes that include the exchanger itself, a family of NHE regulatory factors (NHERFs) that act as a bridge between the exchanger, and a variety of kinases and phosphatases.⁴⁰^,⁴¹ Different stimuli use differing scaffolding complexes to exert their effect. For example, glucocorticoids stimulate Na⁺ absorption and up-regulate NHE3 but not NHE1, 2, or 4, consistent with their respective roles in vectorial transport and housekeeping. Glucocorticoids act via a serum and glucocorticoid inducible kinase, SGK1; SGK1 stimulates the activity of NHE3 by interacting directly with NHERF2.

Alternatively, cAMP inhibits NHE3 by activating protein kinase A, which is recruited to the C-terminus of NHE3 by NHERF1, NHERF2, and an additional cytoskeletal protein, ezrin. In this location, PKA induces its inhibitory effect by phosphorylating NHE3. Cyclic guanosine monophosphate (cGMP) can inhibit NHE3 by triggering the formation of a complex between cGMP-dependent protein kinase II (cGKII) and NHERF2, which anchors cGMP kinase protein (GKAP). Activation of guanylate cyclase C by guanylin or E. coli–stable toxin A increases cGMP content near the brush border to locally activate cGKII (Fig. 99-7), which then inhibits NHE3 activity.⁴⁰