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

Figure 99-7. Second messengers: cAMP and cGMP. Five steps are involved in the transduction of an external signal into a change in cellular function: (1) Binding of either a stimulatory or an inhibitory agonist to an appropriate receptor of the membrane-bound adenylate cyclase or guanylate cyclase system. (2) Binding of ligand to receptor, modulates the cyclase activity either within the same molecule in the case of guanylate cyclase, or by activating the corresponding membrane-bound heterotrimeric guanine nucleotide regulatory proteins (G proteins) in the case of adenylate cyclase. (3) An intracellular signal results from production of cAMP from ATP and cGMP from GTP. (4) Increase in [cAMP]i (intracellular cAMP concentration) activates protein kinases such as PKA and increase in [cGMP]i activates protein kinases such as protein kinase G II, which is fixed to the membrane by myristoylation. Involvement of kinase-anchoring proteins such as A kinase-anchoring proteins (AKAPs) and G kinase-anchoring proteins (GKAPs) has been demonstrated in the signaling. (5) Protein kinase phosphorylation of specific target proteins results in change in the activity of channels or transporters such as chloride channel or the Na⁺-H⁺ exchanger.

In cAMP signaling, binding of stimulatory regulators, such as VIP and prostaglandins, to specific receptors causes activation. Activated receptors couple via G_s to signal adenylate cyclases to catalyze the conversion of ATP to cAMP, which then activates specific cAMP kinases. An inherent GTPase returns G_s to its nascent state; in cholera, the toxin prevents this occurrence by covalently modifying G_s, leaving enterocyte turnover as the only recourse to returning the tissue to its basal state. Other hormones, such as somatostatin, trigger the activation of inhibitory G proteins (G_i) to decrease cAMP. The adenylate cyclase cascade is localized to the basolateral membrane of epithelial cells.

In cGMP signaling, cGMP is generated by the activation of membrane or soluble guanylate cyclases (GCs). In contrast to the adenylate cyclases, membrane GCs are single-pass transmembrane proteins for which the extracellular domain serves as the receptor-binding domain and the intracellular domain catalyzes conversion of GTP to cGMP. Thus, the GCs are specific for their ligands, which include the endogenous atrial natriuretic peptides, guanylin and uroguanylin, as well as enterotoxins such as the heat-stable enterotoxin of E. coli. The intestinal cGMP protein kinase (PGII) is tethered to the membrane via a myristoylated N-terminal region. The soluble GCs are the target of nitric oxide (NO) activation; they are minimally expressed in the small intestinal epithelium, but they are present in colonic epithelia, subepithelial elements, and smooth muscle, where they cause muscular relaxation. ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; G_i, inhibitory G protein; G_s, stimulatory G protein; GTP, guanosine triphosphate; PKA, protein kinase A; PKG, protein kinase G; STa, heat-stable toxin; VIP, vasoactive intestinal peptide.

ELECTRONEUTRAL SODIUM CHLORIDE ABSORPTION

Sodium absorption is coupled to the movement of Cl⁻ through a Cl⁻-HCO₃⁻ anion exchanger, located specifically in the ileum and proximal colon. The rates of the transporters are similar and coordinated by cell pH and HCO₃⁻. Alkalinization of the cell by NHE drives the exit of HCO₃⁻ in exchange for Cl⁻, resulting in electroneutral Na⁺ and Cl⁻ absorption, maintenance of cell pH, and luminal release of H⁺ + HCO₃⁻ (water and CO₂). The coupling of the two exchangers exhibits species and segmental variations. Thus, it is fairly tight in the ileum and proximal colon, whereas in other intestinal segments, although NHE is dependent on Cl⁻ or HCO₃⁻, the linkage between Na⁺ and Cl⁻ transport is variable. The molecular nature of the anion exchangers are described under HCO₃⁻ transport later.

CHLORIDE (ANION) ABSORPTION

The transepithelial, lumen negative potential difference contributes to the passive movement of Cl⁻ and other anions via the paracellular pathway in the jejunum.⁷ While coupled Cl⁻-HCO₃⁻ and Na-H exchangers govern electroneutral transport in the ileum and proximal colon, sodium-independent Cl⁻-HCO₃⁻ exchange occurs in the distal colon (see Fig. 99-6) (see the discussion of HCO₃⁻ transport, later).

CHLORIDE SECRETION

The principal driving force for the secretion of fluid is the transcellular movement of Cl⁻ from the serosal to the luminal compartment. Na⁺ and water follow passively in response to the ensuing electrical and osmotic gradients (Fig. 99-8). The small and large intestine exhibit a basal rate of Cl⁻ secretion that is maintained by the interplay of cell volume, [Cl]_i, and paracrine, autocrine, neuronal, endocrine, luminal, and immune modulators. Disruptions in the balance of these regulatory processes can lead to secretory diarrhea.

Figure 99-8. Intestinal chloride secretion. Discrete basolateral entry steps and apical exit steps are integral to chloride secretion. The Na⁺-K⁺-2Cl⁻ carrier couples the movement of Na⁺, K⁺, and Cl⁻ in a 1 : 1 : 2 stoichiometric relationship and permits Cl⁻ to accumulate in the cell in a concentration greater than its electrochemical equilibrium. Cl⁻ then exits the cell across the apical membrane by means of a chloride channel; Na⁺ and water follow passively. The Na⁺ and K⁺ that entered with the Cl⁻ are recycled by, respectively, the Na⁺ pump and a basolateral K⁺ channel, both critical to maintaining the driving force. These transporters can be regulated by second messengers such as Ca²⁺, cAMP (cyclic adenosine monophosphate), and cGMP (cyclic guanosine monophosphate).

Several epithelia in the GI tract exhibit electrogenic Cl⁻ secretion. Although there are some tissue-specific regulatory differences, the mechanisms underlying this secretion are remarkably similar. The Na⁺ pump provides the driving force, Cl⁻ enters the cell across the basolateral membrane via an electroneutral cotransporter (NKCC1) that couples the movement of 1Na⁺:1K⁺:2Cl⁻, and Cl⁻ leaves the cell via specific channels on the apical membrane. The Na⁺ entering the cell via NKCC1 exits via the Na⁺ pump, and the K⁺ leaves via K⁺ channels either on the apical or the basolateral membrane. This complex interplay of transporters is an elegant demonstration of cellular economy. The NKCC1 cotransporter effectively moves 2 Cl⁻ and 1 K⁺ uphill for the expenditure of a single Na⁺ ion. The pump-to-leak relation between K⁺ channels and the Na⁺ pump helps to maintain the interior of the cell as electronegative, thereby providing the driving force for Cl⁻ exit. Basolateral K⁺ exit electrically balances the large Cl⁻ flux across the apical membrane. NKCC cotransporters belong to a superfamily of cation transporters and are characterized by their inhibition by the loop diuretics bumetanide and furosemide.⁴²^–⁴⁴ NKCC1 is regulated by many kinases, including a unique PASK (proline-alanine–rich STE20-related kinase), phosphatases, actin-myosin interactions, cell volume, and intracellular Cl⁻.

At least three classes of Cl⁻ channels, belonging to distinct protein families and with distinct electrophysiologic characteristics, have been identified in secretory epithelia. The most important of these is the cystic fibrosis transmembrane conductance regulator (CFTR), which is coded for by the gene that is defective in cystic fibrosis. The crucial role of this channel is underscored by the exocrine pathologies that are the hallmark of cystic fibrosis. In addition to abnormalities of the lungs, sweat glands, and pancreas, infants with cystic fibrosis often present with meconium ileus, and 15% of adults with cystic fibrosis exhibit distal intestinal obstructive syndrome. Interestingly, the major pathology of the CFTR-deficient mouse is meconium ileus, which results in early mortality unless treated with an osmotic laxative.

CFTR has been localized to the apical membrane of various segments of the small and large intestine, with greater expression in the crypts than the villus or surface cells.⁴⁵ CFTR is a 250-kd membrane protein belonging to the superfamily of ATP binding cassette proteins. CFTR is a small-conductance (8-10 pS) linear channel, with an ion selectivity of Br⁻ > Cl⁻ > I⁻ > F⁻ that also can transport HCO₃⁻.⁴⁶^,⁴⁷ It has two membrane-spanning domains, two ATP-binding domains, and a regulatory (R) domain that has many consensus sequences for phosphorylation by kinases, specifically for PKA. Gating of the channel is regulated by sequential binding of ATP to the two domains and phosphorylation and dephosphorylation of the R domain. In secretory diarrhea such as cholera, PKA activates the R domain to increase channel activity while simultaneously stimulating the recruitment of CFTR-bearing endosomes to the apical membrane, increasing the number of channels (Fig. 99-9). Although more than 1000 mutations of CFTR have been identified, approximately 70% of cystic fibrosis patients carry the ΔF508 mutation, a single amino acid deletion that results in improper folding and diversion of the protein to the endoplasmic reticulum-associated degradative pathway rather than to the apical membrane. The protein is pleiotropic and interacts with a variety of other proteins, influencing their expression and regulation, and being modulated, in turn, by mechanisms that are not fully deciphered. Some of this interaction can occur through crosstalk of CFTR with scaffolding proteins it shares with other transporters; for example, the C-terminal of CFTR interacts with NHERF, a protein that also binds to NHE3.

Figure 99-9. Mechanism of action of Vibrio cholerae enterotoxin. V. cholerae produces an enterotoxin, and a zonula occludens toxin (ZOT) that disrupts tight junction permeability, and other toxins (not shown here) that are not fully identified. CT induces diarrhea by inhibiting the absorptive processes in the villus and surface epithelial cells (A) and by stimulating the secretory processes in the crypt epithelial cells (B). As shown in A, a CT-mediated increase in cAMP leads to an inhibition of salt absorption (Na⁺-H⁺ and Cl⁻-HCO₃⁻ transporters), but does not affect Na-glucose transport. The events that follow enterotoxin binding leading to cAMP generation are similar in villus and crypt cells. As shown in B, the enterotoxin binds to ubiquitous GM1 gangliosides via its B subunits on the intestinal brush border membrane (1) and, by capturing elaborate cellular processes, including lipid rafts, retrograde endocytosis, and endoplasmic reticular proteolysis (2), transmits the A1 peptide of its A subunit to the basolateral membrane. At the basolateral membrane, A1 catalyzes the adenosine diphosphate ribosylation of Gα_s (G_s). This permanently activates G_s by covalently inhibiting the nascent GTPase and preventing activated G_s from returning to its nascent state (3). Activated G_s then stimulates adenylate cyclase to produce an unregulated increase in cAMP (4). Although the major pathophysiologic effects are attributed to the enterotoxin, V. cholerae also recruits multiple components of the PINES, including enteric neurons, enterochromaffin cells, prostaglandins, and serotonin (as shown at the bottom of the figure) that together contribute to the copious fluid output in the small intestine. In addition, interneurons between the small and large intestine underlie the ability of cholera toxin in the small intestine to trigger a reflex secretory response in the colon. AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; CT, cholera toxin; EC, enterochromaffin; GM1, monosialotetrahexosylganglioside; G_s, stimulatory G protein; 5-HT, serotonin; PG, prostaglandin; PINES, paracrine, immunologic, neural, and endocrine systems; VIP, vasoactive intestinal polypeptide.

Diminished intestinal fluid secretion in CF^−/− mice is associated with goblet cell hyperplasia, increased crypt cell proliferation, Paneth cell abnormalities, and increased inflammation.⁴⁸ Whether all these changes are secondary to decreased fluid secretion or result from more direct effects of CFTR on other protein function remains to be determined (see later). By combinatorial chemistry, a series of compounds have been designed to specifically inhibit or stimulate CFTR, thereby serving as therapeutic strategies for secretory diarrhea or cystic fibrosis.⁴⁹

CHLORIDE CHANNELS

CIC Family Channels

Chloride channels of the distinct ClC family,⁵⁰^,⁵¹ in particular the widely distributed plasma membrane protein ClC2, are of clinical interest as a potential salvage pathway for Cl⁻ transport in cystic fibrosis patients and as the specific target of lubiprostone, a novel laxative.⁵² ClC2 is involved in regulation of epithelial transport, intracellular pH, intracellular chloride and cell volume. Because of significant differences in its localization in a diverse set of human and animal models, however, its exact role in chloride secretion is uncertain. Thus, although lubiprostone activates ClC2 in the apical membrane of the human colon carcinoma cell line T84, it cannot rescue Cl⁻ secretion in the CFTR-deficient mouse. This dichotomy suggests that the channel is located on the basolateral membrane of the CF mouse. Further research will be needed to clarify how these channels and their pharmacologic agonist work in regulating chloride secretion in humans.

CALCIUM-ACTIVATED CHLORIDE CHANNELS

A third class of Cl⁻ channels, the calcium-activated Cl⁻ (CLCA) channels, are involved in the diarrhea of rotavirus infection. The rotavirus toxin NSP4 activates calcium-dependent Cl⁻ secretion, especially in the colons of young mammals. Despite an understanding of the regulation of calcium-dependent Cl⁻ secretion in many epithelia, however, the molecular nature of these channels has eluded precise identification; recent evidence points to a group of proteins, the bestrophins, as potential candidates. It remains to be determined if bestrophins are regulated channels per se or regulators of channels or whether they use auxiliary proteins to function as channels.⁵³^,⁵⁴ The CLCA proteins also might play a role in goblet cell function: Mouse CLCA3 is severely reduced in the CF^−/− mouse, and its restoration ameliorates the severity of intestinal cystic fibrosis disease.⁵⁵

POTASSIUM TRANSPORT

A plethora of K⁺ transport processes help the intestine cope with its need to balance fluid and electrolyte movement.⁵⁶^,⁵⁷ Potassium secretion and absorption occur along the length of the intestinal tract, although the specific pathways are segment specific. In the small intestine, the luminal negative potential difference drives the passive absorption of K⁺. In contrast, K⁺ absorption in the distal colon occurs by primary active transport via H⁺,K⁺-ATPase pumps located on the luminal membrane. These H⁺,K⁺-ATPase pumps are P-type ATPases, related to the gastric H⁺,K⁺-ATPase, and least two colonic isoforms have been identified: a ouabain-sensitive isoform in the crypt cells and a ouabain-insensitive isoform in the surface cells. Depletion of aldosterone and K⁺ upregulates the ouabain-insensitive H⁺,K⁺-ATPase and stimulates K⁺ absorption.

Potassium channels are the largest group of ion channels in the human genome. A number of K⁺ channels have been localized to intestinal cells, but only a few are mentioned here (using the nomenclature of the Human Genome Organization). Found primarily on the basolateral membrane of intestinal epithelial cells, K⁺ channels contribute significantly to intestinal electrolyte homeostasis through several mechanisms. K⁺ channels modulate the hyperpolarization of the cell interior that is needed for vectorial, voltage-driven transport processes. In the basolateral membranes of the small and large intestine, the cAMP-activated KCNE3/KCNQ1 channels and the Ca²⁺-calmodulin–activated KCNN4 channels hyperpolarize the membrane and promote Cl⁻ secretion. Basolateral K⁺ channels contribute to the transepithelial potential difference, which influences paracellular movement; K⁺ secretion in the colon occurs via apical KCNMA1 (MaxiK) channels, which are regulated by mineralocorticoids. In response to cell swelling, K⁺ channels are activated and cause a regulatory volume decrease, a critical function for intestinal cells faced with constant fluctuations in osmolarity. K⁺ channels also play roles in differentiation, apoptosis, and carcinogenesis, functions that involve different channel proteins and that can be regulated by a number of processes including protein modifications (phosphorylation, sumoylation), membrane voltage, cytosolic calcium, pH, cell swelling, and cell metabolism.

BICARBONATE TRANSPORT

Bicarbonate is a metabolic product and a critical anion in fluid homeostasis in the intestine. In clinically significant diarrhea, bicarbonate is the major anion in the stool. It is secreted by electrogenic and electroneutral processes in the duodenum, ileum, and colon. Being a metabolic product, intracellular HCO₃⁻ can arise from intracellular metabolism, diffusion of CO₂ or the action of transporters such as the basolateral Na⁺-HCO₃⁻ cotransporter. Electrogenic HCO₃⁻ secretion can occur via apical anion channels, including CFTR; however, the major mechanism for HCO₃⁻ secretion in the small and large intestine is inexorably linked to the inward movement of Cl⁻ and occurs through apical Cl⁻-HCO₃⁻ exchangers.⁵⁸ It is postulated that electrogenic Cl⁻ secretion via CFTR provides luminal Cl⁻, which is then recycled across the apical membrane in exchange for intracellular HCO₃⁻. SCFA-dependent bicarbonate secretion also has been observed in surface cells of the colon.⁵⁹

Although the prototype of anion exchangers, the red cell Cl⁻-HCO₃⁻ exchanger (AE1), has been extensively studied, identification of intestinal exchangers is relatively recent. In a reorganization of nomenclature, the more than 360 identified solute carriers (SLC) are now classified in one of 46 families; of these, the SLC4 bicarbonate transporter family encompasses the red cell anion exchanger (AE1) and its epithelial isoforms (e.g., AE2, AE3, and AE4).⁶⁰^–⁶² The precise role of the AEs in overall intestinal Cl⁻-HCO₃⁻ transport remains to be clarified, and apical and basolateral localizations have been suggested.

Structurally distinct from the SLC4 bicarbonate transporter family is the SLC26 multifunctional anion exchange family. SLC26 exchangers can transport Cl⁻, HCO₃⁻, sulfate, formate, oxalate, hydroxyl ions, and other anions with differing affinities. Their varied distribution along the GI tract provides them with the flexibility to handle a variety of luminal anions. Two members of this family, SLC26A3 and SLC26A6, are of special interest.¹⁰^,⁶³^–⁶⁵ The former, first identified as DRA (down-regulated adenoma), is expressed abundantly on the apical membranes of colonocytes, but not enterocytes, and transports more than 2Cl⁻:1HCO₃⁻ ion. Mutations in DRA cause congenital chloride diarrhea, which manifests with severe diarrhea, volume depletion, and metabolic alkalosis. In contrast, SLC26A6, also known as the putative anion transporter 1 (PAT1), is expressed abundantly in the apical membrane of villus enterocytes and less so in the colon. PAT1 transports more than 2HCO₃⁻:Cl⁻ ion; a separate HCO₃⁻ conductive pathway might mediate bicarbonate secretion into the duodenum.

In contrast to the apical NHE isoforms, anion exchangers are present on the apical membranes of crypt and surface cells. A number of regulatory pathways, including cGMP-mediated pathways, strongly influence HCO₃⁻ secretion. The physiologic implications of the spatial distribution and varied anion transporters in net HCO₃⁻ secretion remain to be elucidated. It is, however, intriguing that HCO₃⁻ transport is dependent on CFTR and that anion exchange processes are down-regulated in cystic fibrosis.

SHORT-CHAIN FATTY ACID TRANSPORT

The 2- to 4-carbon short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are generated by the fermentative action of the bacterial microflora on poorly absorbed carbohydrates. Unlike the small intestine, in which Cl⁻ and HCO₃⁻ predominate, the major anions (60 to 150 mmol/kg) in the colon are SCFAs; thus, there is a large SCFA concentration gradient across the colonic epithelium. The magnitude of the daily colonic load and absorption of SCFAs is comparable to that of colonic Na⁺. SCFAs are a major source of metabolic fuel for the colonocytes, modulate epithelial growth and differentiation, and have been implicated in the pathogenesis of and therapy for several inflammatory diseases of the colon, such as bypass colitis.

SCFAs are rapidly absorbed in the colon and also greatly enhance Na⁺ and fluid reabsorption through linked transport mechanisms and by upregulating the expression of NHE3 on the apical membrane of colonocytes.⁶⁶ SCFAs are weak electrolytes and can be ionized or protonated. Ionized SCFAs need specific carriers, whereas nonionized protonated species can diffuse across the colonocyte membrane; at luminal colonic pH, SCFAs are 95% to 99% ionized.

A picture of the molecular basis of SCFA transport is beginning to emerge.³²^,⁶⁷ First, apical NHEs can create an acidic pH microclimate and enhance the diffusion of protonated SCFAs into the cell. Monocarboxylate transporters (MCTs), members of two different SLC families, are involved in electroneutral carrier-mediated SCFA transport. Members of the SLC16 family, specifically SLC16A1 (MCT1), transport 1H:1SCFA and require an ancillary protein for their function.⁶⁸ High-affinity (SLC5A8) and low-affinity (SLC5A12) Na⁺-dependent MCTs have been identified in the colon and intestine. Although their molecular identity is unclear, Cl⁻-butyrate exchangers and SCFA-HCO₃⁻ exchangers, functionally coupled to Na⁺-H⁺ exchange, might account for SCFA promotion of electroneutral Na⁺ and Cl⁻ absorption.⁵⁹^,⁶⁹

PARACRINE IMMUNONEUROENDOCRINE SYSTEM

Extracellular factors from classic paracrine, immunologic, neural, and endocrine systems (PINES) as well as autocrine factors and luminal content regulate intestinal ion transport. The borders separating members of PINES are arbitrary at best, because there is considerable overlap and crosstalk of the underlying factors, and patterns can be altered in disease states (Fig. 99-10). Luminal mechanical (stroking and stretch) or chemical (toxins) stimuli can activate mechanoreceptors and chemoreceptors, respectively, to activate one or more arms of PINES. All these interactions are compounded by many of the factors acting through cell-specific multiple receptors and signaling pathways.

Figure 99-10. Model depicting the integral components of the enteric nervous system and the immune system that regulate intestinal ion transpart. The components include (1) neurons responsive to intraluminal mechanical and chemical stimuli (e.g., food, bile acids, bacterial toxins, rotavirus); (2) interneurons in either the myenteric or submucosal plexuses; (3) secretory neurons that release acetylcholine (Ach) which acts on epithelial cells; and (4) interactions among secretory neurons and blood vessels, immune cells, and paracrine cells. Intestinal cells can release an array of secretory factors, which can act either directly on the epithelium or indirectly by stimulating the mesenchymal cells or enteric neurons to release prostaglandins (PGs) or acetylcholine (Ach). See text for details.

Within the subepithelium, structural elements of PINES, including blood vessels, are in close proximity (see Fig. 99-10), so release of mast cell mediators can easily target neurons and vice versa; this proximity and interplay contributes to the minute-by-minute local regulation necessary in the intestine. Although it is possible to separate the specific effects of an individual component in vitro, clinically they are inextricably intertwined. For example, Verner-Morrison syndrome (pancreatic cholera) is classified as an endocrine-mediated diarrhea, because pancreatic islet cell tumors produce large amounts of vasoactive intestinal peptide (VIP). In the healthy adult, however, VIP is not found in the pancreas but is a peptidergic neurotransmitter in the enteric nervous system that stimulates epithelial cell secretion and smooth muscle relaxation. Alternatively, a single agonist such as cholera toxin directly acts on epithelial cells while simultaneously stimulating neural, paracrine, and immune responses. In a third example, serotonin (5-hydroxytryptamine [5-HT]) released by mucosal enterochromaffin cells acts via distinct receptors on epithelial cells to directly stimulate secretion; it acts on myenteric neurons to release acetylcholine (Ach) and elicit migratory contractions; or it acts on submucosal neurons to release Ach and calcitonin gene-related peptide to stimulate peristalsis and secretory reflexes.

Fluid secretion is the major component in the production of diarrhea, and the organism’s defensive response to intestinal challenge; motility, mucus secretion, and blood flow, all regulated by PINES, are important adjuncts to the process. The involvement of PINES in motility helps to explain diarrhea associated with rapid intestinal transit (e.g., following gastrectomy), altered anorectal motility (e.g., small-volume diarrhea), or decreased motility (e.g., bacterial overgrowth). Decreased motility leads to a increase in the bacteria in the small intestine, which causes diarrhea by a variety of mechanisms (see Chapters 97 and 102). Alternatively, inflammatory mediators such as prostaglandins or bacterial enterotoxins target both the epithelial and muscle layers to elicit a coordinated secretory response, while promoters of absorption such as opiates and enkephalins suppress motility and promote electrolyte absorption. Thus, PINES allow for a coordinated and integrated response to multiple extracellular signals.

EXTRACELLULAR REGULATION

Tables 99-1, 99-2 and 99-3 list the major neurohumoral substances and toxins that modulate intestinal fluid transport. Agents that promote net fluid secretion generally inhibit Na⁺ absorption and stimulate Cl⁻ secretion, whereas agents that promote net fluid absorption increase Na⁺ uptake and attenuate Cl⁻ secretion. In a healthy person, net absorption prevails, and when this balance is disrupted, diarrhea can ensue. It is unclear if there is a corollary for a predominant absorptive pattern in a subset of patients with constipation.

Table 99-1 Agents That Stimulate Intestinal Absorption of Fluid and Electrolytes

Endogenous Absorbagogues

α-Adrenergic agonists

Aldosterone

Angiotensin

Enkephalins

Glucocorticoids

Growth hormone

Neuropeptide Y

Peptide YY

Prolactin

Short-chain fatty acids

Somatostatin

Pharmacologic Agents

Berberine

Clonidine (α₂-agonist)

Cyclooxygenase inhibitors

Glucocorticoids

Lithium

Mineralocorticoids

Octreotide

Opiates

Propranolol

Table 99-2 Endogenous Agonists of Intestinal Secretion

AGONIST	INTRACELLULAR MEDIATOR	SOURCE
Acetylcholine	Ca²⁺	ENS
Adenosine	cAMP	Immune cells
Arachidonic acid	cAMP	Immune cells, cell membranes
Atrial natriuretic peptide	cGMP	Heart
Bombesin	Ca²⁺	??
Bradykinin	cAMP	Immune cells
Calcitonin, calcitonin gene-related peptide	?	ENS
Galanin	Ca²⁺
Gastric inhibitory polypeptide	?	??
Gastrin	Ca²⁺(PKC/MaPK?)	Endocrine cells
Guanylin	cGMP	Goblet, epithelial cells
Histamine	Ca²⁺	Immune cells
Leukotrienes	?	Immune cells
Motilin	Ca²⁺	Endocrine M cells
Neurotensin	Ca²⁺	ENS
Nitric oxide	cGMP	Immune cells, mesenchymal cells
Peptide histidine isoleucine	cAMP	ENS
Platelet activating factor	cAMP	Immune cells
Prostaglandins	cAMP	Immune cells, mesenchymal cells
Reactive oxygen metabolites	cAMP	Immune cells
Secretin	cAMP	Endocrine cells
Serotonin	Ca²⁺	ENS
Substance P	Ca²⁺	ENS
Vasoactive intestinal polypeptide	cAMP	ENS

cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; ENS, enteric nervous system.

Table 99-3 Luminal Agents That Stimulate Intestinal Secretion

AGENT	INTRACELLULAR MEDIATOR
Bacterial Enterotoxins
Aeromonas sp.	cAMP
Campylobacter jejuni	cAMP
Clostridium difficile (toxin A)	Ca²⁺
Clostridium perfringens	??
Escherichia coli (heat labile toxin)	cAMP
E. coli (heat stable toxin)	cGMP
Rotavirus NSP₄	Ca²⁺
Salmonella sp.	cAMP
Vibrio cholerae
accessory cholera enterotoxin	??
enterotoxin	cAMP
zona occludens toxin	??
Vibrio parahaemolyticus	Ca²⁺
Yersinia enterocolitica	cGMP
Miscellaneous Agents
Bile salts	cAMP/Ca²⁺
Laxatives	??
Long-chain fatty acids	cAMP/Ca²⁺

cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; NSP, nonstructural protein.

ENDOCRINE, PARACRINE, JUXTACRINE, AND AUTOCRINE REGULATION

Intestinal transport is modulated by classic endocrine, paracrine, juxtacrine, and autocrine processes. Intestinal endocrine cells are interspersed between epithelial cells and function as sensors that rapidly respond to changes in the luminal environment by releasing secretory granules containing biogenic amines and hormones; these mediators cross the basolateral membrane. These hormones can act either in a classic endocrine manner on distant target cells (via the circulation) or in a local (paracrine) manner by affecting neighboring cells in the intestinal wall. Juxtacrine mediators are those released from nonendocrine cells, such as neural and inflammatory cells, and they affect neighboring cells. Intestinal mesenchymal cells, in particular myofibroblasts, are a rich source of cytokines, chemokines, eicosanoids, and growth factors that can alter intestinal transport. Epithelial cells can self-regulate (autocrine) by secreting factors such as eicosanoids, which act on epithelial cell receptors to alter function.⁷⁰

NEURAL

Neural input is critical in the regulation of fluid and electrolyte transport (see Fig. 99-10) and involves interactions of the parasympathetic and sympathetic divisions of the autonomic nervous system with the labyrinthine enteric nervous system (ENS) (see Chapters 1, 97, 98 and 120). Cholinergic stimulation of secretion, predominantly through parasympathetic vagal input, and adrenergic stimulation of absorption through prevertebral and sympathetic ganglia have long been recognized as fundamental neural pathways affecting the intestinal epithelium. The ENS is the end controller of neural activity in the intestinal wall, however, independently integrating the regulation of the epithelia, muscles, and blood vessels, with input and modification from the central nervous system.

Like other neural networks, the ENS has reflexes that can have important clinical implications. Target cells for neurons include components of PINES, blood vessels, and, of course, epithelial cells. Sensory input into the ENS comes from changes in the luminal content (e.g., acidity, dietary content, pathogens) or volume (e.g., stretch). Thus, acid or distension can activate TRVP1 vanilloid receptor on capsaicin-sensitive afferent nerves, which in turn evokes secretion by stimulating submucosal neurons and causes vasodilation by directly activating submucosal arterioles.⁷¹^,⁷² Endocrine, dendritic, and/or paracrine cells releasing serotonin, adenosine (see later), and other signals are implicated as auxiliary sensors. Primarily cholinergic, interneurons are believed to underlie the ENS-mediated regulation of colonic epithelial responses to distant small intestinal challenges. Motor neurons that innervate epithelial and submucosal cells can be cholinergic or VIPergic, each releasing additional neuroactive substances.

A basal cholinergic secretory drive is tempered by the sympathetic tone; loss of adrenergic sympathetic innervation in diabetic neuropathy is associated with the development of “diabetic diarrhea” and may be corrected by α₂-adrenergic agonists.⁷³ As in the brain, the number and variety of compounds that act as neuroactive agents has ballooned, and this has added to the complexity of our elucidating the underlying mechanisms. Thus, neurons can release specific combinations of mediators, such as VIP, cholecystokinin, gastrin-releasing peptide, and ATP, rather than a single substance. Individual neurotransmitters can have biphasic effects varying with concentrations. Furthermore, agents can act as classic neurotransmitters; alternatively, they can act as neuromodulators, fine-tuning the neuronal circuits of presynaptic sites of neurons, or, as in the case of serotonin, they can function as paracrine substances.

IMMUNOLOGIC

The clinical correlation between intestinal inflammation and diarrhea is obvious (see Chapter 2), with ulceration, exudation of protein, changes in motility, and loss of absorptive surface area implicated as causing the fluid losses of (inflammatory) diarrheas. Immunocompetent cells of the intestine reside chiefly in the lamina propria, form the gut-associated lymphoid tissue (GALT), and often are distinct from their systemic counterparts; in noninflamed intestine, T lymphocytes account for 60%, with smaller numbers of B lymphocytes and plasma cells (25% to 30%), macrophages (8% to 10%), mast cells, and polymorphonuclear cells (2% to 5%) (usually eosinophils).⁷⁴ These cells secrete a vast array of soluble products (chemokines, cytokines, eicosanoids, nucleotides, biogenic amines) whose actions are intimately intertwined with other aspects of PINES. Intestinal inflammation increases the number of immunocytes, the cause of the inflammation determining the type of inflammatory cells; for example, acute bacterial infections increase polymorphonuclear leukocytes, whereas parasitic infections dramatically enlarge the mast cell population and celiac disease is characterized by intraepithelial lymphocytes. In IBD, there is activation of all components of GALT with an increase in immunoglobulin (Ig)G-secreting cells.⁷⁵^,⁷⁶ Thus, the cause of the inflammatory reaction can determine the type of immunocytes recruited, the range of cytokines released, and the specific effects on transport and motility.

Many inflammatory mediators are potent secretagogues, including peptides (e.g., cytokines, platelet-activating factor, substance P, interferon gamma, kallikreins, and bradykinin), eicosanoids (e.g., arachidonic acid, leukotrienes, and prostaglandins), and oxidants (e.g., superoxides). These mediators either interact directly with epithelia to alter ion transport and barrier function or elicit these effects indirectly by activating other PINES elements (see Fig. 99-10). Prostaglandins are central to the secretory response associated with inflammation, affecting various PINES components, such as enteric neurons, and with mediators such as bradykinin, which liberates arachidonic acid to stimulate prostaglandin production. In interpreting the effects of the inflammatory mediators in normal model systems, it is important to recognize that in vivo, cells damaged by the inflammatory process might not be able to function normally. With this caveat, a few examples are provided.

Mucosal mast cells are strategically located in close proximity to enteric neurons, blood vessels, and epithelial cells, and they are central to several inflammatory reactions. Mast cell mediators including histamine, eicosanoids, and cytokines elicit secretion by direct effects on the epithelial cells and by indirect neural stimulation and prostaglandin release.

The mechanisms of secretion resulting from polymorphonuclear infiltration of the mucosa recently have become better understood. Polymorphonuclear leukocytes, responding to chemoattractants (e.g., fMLP [formyl-Met-Leu-Phe]), uniquely present in the inflamed colon, leave the vasculature and interact with epithelial cells.⁷⁷ The white cells burrow through the intercellular space of the colonic cells in a complex integrin-dependent process. The migrating leukocytes release 5-AMP, which is converted to adenosine by apical membrane enzymes. Adenosine is a potent secretagogue, and this adenosine-stimulated secretion might serve as a mechanism to cleanse the crypt lumen.⁷⁸ Thus, complex specific immunocyte-epithelial cell interactions are important in alterations of electrolyte secretion associated with mucosal inflammation. Anti-integrin targeted therapy may be effective in treating Crohn’s disease (see Chapter 111).

During inflammation, oxidants such as superoxides, hydrogen peroxide, and hydroxyl radicals released from neutrophils stimulate Cl⁻ secretion; cytokines such as interleukin-1 and interleukin-3 also stimulate secretion.⁷⁹ In contrast, interferon-γ and TNF-α can cause diarrhea more through an antiabsorptive effect, by down-regulating particular transporters ⁸⁰^–⁸² or by altering permeability of tight junctions. Similarly, studies on colonic specimens from patients with IBD or ulcerative colitis indicate that diarrhea in these entities results not from stimulated secretion but from increased tissue permeability combined with decreased apical Na⁺ channel, basolateral K⁺ channel, and Na⁺,K⁺-ATPase expression, which decreases NaCl reabsorptive capacity.⁷⁶ The multiplicity of transport malfunctions seen in IBD might reflect a sick cell syndrome rather than specific alterations modulated by one or two unique cytokines.

SYSTEMIC EFFECTS

Acid-base balance modulates intestinal electrolyte transport in vivo and in vitro. Changes consistent with metabolic acidosis are potent stimulators of electroneutral NaCl absorption, whereas metabolic alkalosis inhibits this process.⁸³^–⁸⁴ Intracellular bicarbonate concentrations can modulate basal Cl⁻ secretion, and intracellular pH and Pco₂ can alter Na⁺:H⁺ exchange. Volume status and intestinal blood flow also alter ion transport. Any decrease in intravascular volume, such as with hemorrhage, elicits a series of responses that increase fluid absorption. Cardiopulmonary mechanoreceptors and carotid baroreceptors increase sympathetic input into the ENS, resulting in decreased secretion. Angiotensin II, antidiuretic hormone, and atrial natriuretic peptide also can contribute to regulation of intestinal fluid transport in these conditions.⁸⁵^–⁸⁶ The metabolic status of the intestine has an impact on its transport capability; a well-fed intestine transports more effectively.⁸⁷ There also are segmental preferences for metabolic fuels. Although the entire intestinal tract uses glucose, the small bowel effectively uses glutamine, and the colon preferentially uses SCFAs, particularly butyrate.⁸⁸^,⁸⁹

OSMOTIC EFFECTS

Unlike the kidney, the intestinal epithelium cannot maintain an osmotic gradient. Under normal physiologic conditions, the duodenum and upper jejunum are subject to major fluid shifts as they adjust to dietary intake of hypertonic foods and liquids. Rapid equilibration usually is accomplished by movement of water into the intestinal lumen, and absorptive processes along the remainder of the intestine steadily decrease the luminal volume. The continued presence of a nonabsorbable solute within the intestinal lumen, however, can negate functioning absorptive pathways in the distal intestine. This is the basis for osmotic diarrhea (see Chapter 15).

Carbohydrates, usually disaccharides, are a common source of nonabsorbable solute. Disaccharides must be hydrolyzed to monosaccharides before they can cross the apical membrane of the small intestine, (see Chapters 100 and 101). The most common clinical example of maldigestion resulting from a deficiency of a specific disaccharidase is lactose intolerance, in which the glucose-galactose disaccharide cannot be broken down because of lactase deficiency. Because the human intestine does not naturally possess a lactulase, the disaccharide lactulose reliably increases small intestinal fluid because of luminal hyperosmolarity and bacterial fermentation of the disaccharide. The limited intestinal absorptive capacity for several sugars found in processed foods and drinks, such fructose and sorbitol, can play important, but often overlooked, roles in osmotic diarrhea, bloating, abdominal pain, and irritable bowel syndrome.

The physiology of carbohydrate-induced osmotic diarrhea is complicated by the fact that nonabsorbable solutes in the small bowel can be converted into absorbable solutes by colonic bacteria. Almost all classes of carbohydrates not absorbed by the small intestine are rapidly converted to SCFAs once they cross the ileocecal valve and encounter the colonic bacterial flora; these SCFAs can be absorbed and serve as metabolic fuel for the colon. Thus, depending on the rate of carbohydrate conversion to SCFAs and the colonic capacity for SCFA absorption, small intestinal fluid loss may be compensated by colonic fluid absorption. However, if the capacity of the intestinal flora to convert carbohydrates to SCFAs is maximized, or if the SCFA absorptive capacity of the colon is exceeded, additional unmetabolized carbohydrates could pass through the colon and exacerbate the osmotic effects of nonabsorbable solute.⁹⁰

Cations (e.g., magnesium) or anions (e.g., sulfate and phosphate) are absorbed poorly by the normal intestine. Increased ingestion of these ions easily leads to osmotic diarrhea, which may be intended, such as with laxatives, or unintended, such as with ingestion of magnesium-containing antacids or supplements.

In clinical situations in which there is malabsorption or a generalized destruction of the epithelium, solutes normally absorbed readily can remain in the intestinal lumen and thereby contribute an osmotic component to an inflammatory diarrhea or a malabsorptive state. Osmolality is an important factor in patients receiving enteral nutrition (see Chapters 4 and 5). For example, complex carbohydrates provide a significant amount of calories with minimal osmolality compared with simple sugars. Absorption of dipeptides and tripeptides instead of amino acids reduces intestinal osmolality. This balance between calories and osmolality becomes clinically relevant in effectively designing appropriate tube-feeding regimens. Osmolality also is an important factor in designing second-generation oral rehydration therapy (ORT) formulations; by replacing glucose with complex carbohydrates such as rice, intestinal absorption is stimulated further by creating a hypotonic luminal environment, thereby enhancing water absorption. In addition to providing numerous sugar molecules per milliosmole, complex carbohydrates such as rice and amylase-resistant starches have another advantage in ORT: They are metabolized by commensal bacteria in the colon to release SCFAs, which in turn promote fluid absorption (see the earlier discussion of SCFAs).⁹¹

SPECIFIC REGULATORY FACTORS

ABSORPTIVE

Intestinal agents that stimulate absorption are listed in Table 99-1.

Mineralocorticoids (e.g., aldosterone) primarily affect electrogenic Na⁺ absorption in the distal colon and have little effect on the small intestine, which exhibits electroneutral Na⁺ absorption. Aldosterone increases the activity and numbers of the apical membrane ENaC (see the earlier discussion of Na channels) and stimulates activity of the Na⁺/K⁺ pump and SGK1, resulting in an increase in Na⁺ absorption. Aldosterone increases K⁺ absorption and K⁺ secretion.⁹² Neonates exhibit a correlation between high circulating levels of aldosterone and enhanced colonic Na⁺ absorption. Clinically, the physiologic role of aldosterone can be seen in the increased colonic absorption after Na⁺ depletion (aldosterone stimulation) or in the diarrhea associated with Addison’s disease (aldosterone deficiency).

Glucocorticoids are also potent stimulators of Na⁺ absorption in the small intestine and the colon, in addition to having well-documented anti-inflammatory effects. At low concentrations, glucocorticoids stimulate electroneutral Na⁺ absorption and suppress electrogenic Na⁺ absorption, whereas at high concentrations they stimulate both processes. The actions of glucocorticoids are complex and are species and segment specific, and they may be directed at the level of apical Na⁺ transporters and at the Na⁺ pump. For example, in the rabbit jejunum, ileum, and colon, glucocorticoid-stimulated increases in Na⁺ absorption are associated with selective increases in NHE3 but not NHE2 or NHE1 mRNA and protein. Both glucocorticoids and aldosterone evoke rapid cellular responses involving the SGK1 pathway as well as genomic transcriptional effects. These effects might account, in part, for the potent antidiarrheal action of glucocorticoids in a wide variety of clinical settings.³⁸

Catecholamines, enkephalins, and somatostatin all stimulate electroneutral Na⁺ absorption and often decrease HCO₃⁻ secretion with similar patterns of action. They bind to specific heptahelical membrane receptors and activate the Gα_i cascade, which in turn suppresses the prosecretory cAMP signaling cascade.

Catecholamines (e.g., dopamine and epinephrine) acting as α-adrenergic agonists, all have similar absorptive properties. The theoretical basis for the use of clonidine as an antidiarrheal agent, particularly in diabetic diarrhea, is rooted in this adrenergic absorptive pathway.⁹³

The use of plant opiates as antidiarrheal agents dates back two millennia to the early Egyptians and underscores their effectiveness. Elucidating their therapeutic effect led to the characterization of the mammalian opioid peptides—enkephalins, endorphins, and dynorphins—a classic example of molecular mimicry.⁹⁴ Acting via one of three main opioid receptor subtypes—mu, delta, and kappa—the opiates and opioid peptides decrease secretion and promote nonpropulsive motility patterns, thereby increasing transit time. They can act directly on the epithelial and smooth muscle cells or can modify the electrical and synaptic behavior of ENS neurons. The constipation associated with morphine intake can be due to hyperpolarization of secretomotor neurons and suppression of secretion or to a centrally mediated stimulation of sympathetic noradrenergic discharge, or both. Direct activation of K⁺ channels and inhibition of Ca²⁺ channels via a G protein-mediated mechanism underlie these effects. Chronic treatment with opiates leads to tolerance, and diarrhea ensues upon abrupt withdrawal. Clinically, management of constipation in patients receiving opiates as analgesics can be a clinical challenge.

The development of long-acting analogs of somatostatin has transformed this ubiquitously distributed hormone from a physiologically fascinating regulator to a clinically relevant pharmacologic agent (see Chapter 1). In the intestine, enterochromaffin D cells produce somatostatin, which stimulates salt and water absorption in the ileum and colon and blocks the effects of several secretagogues.⁹⁵^,⁹⁶ Somatostatin analogs such as octreotide are effective in treating several types of diarrheal diseases, particularly endocrine-related secretory diarrhea. Their therapeutic effect is due to a combination of actions, including inhibition of hormone release by tumors, slowing of intestinal transit, and a direct effect on epithelial cells. Paradoxically, elevated somatostatin levels, as encountered in somatostatinomas or with large pharmacologic doses of octreotide, can precipitate diarrhea secondary to steatorrhea.⁹³ Other peptide hormones, including peptide YY, angiotensin II, and insulin, have been implicated as proabsorptive agents, but their physiologic significance is as yet unknown.

SECRETORY

Endogenous agents that stimulate secretion are listed in Table 99-2.

Although there are subtle differences in their biologic actions, in general, eicosanoids, including arachidonic acid, prostaglandins, and leukotrienes, inhibit electroneutral NaCl absorption and stimulate electrogenic Cl⁻ secretion.

Most intestinal prostaglandins arise from submucosal immunocytes. They have a major autocrine effect on epithelial cells, but they also modulate enteric nerves and affect intestinal motility and blood flow. Depending on the type of prostaglandin and receptor subtype, prostaglandins primarily act via cAMP and, to a lesser extent, via intracellular Ca²⁺. Prostaglandins can contribute to the basal secretory tone of the epithelium, because cyclooxygenase inhibitors, such as indomethacin or aspirin, increase basal rates of absorption.

Increased intestinal production of eicosanoids contribute to the diarrhea of IBD (see Chapters 111 and 112). Glucocorticoids can decrease prostaglandin synthesis. Although the 5-ASA (acetylsalicylic acid) class of medications, which are a mainstay of the treatment of IBD, target cyclooxygenase and decrease prostaglandin production, their clinical efficacy probably depends on additional mechanisms of action, including effects on other inflammatory pathways, including nuclear factor κB (NFκB) and reactive oxygen species (ROS).⁹⁷^,⁹⁸ Leukotrienes are considered also to have a role; the mechanism of their action is less well understood but might involve activation of secretomotor neurons in the subepithelium.

Acetycholine, serotonin, guanylin, VIP, and other hormones and neurotransmitters have been implicated as stimulators of intestinal secretion, generally acting to inhibit electroneutral NaCl absorption and stimulate Cl⁻ secretion. They are classified by their mechanisms of action. Acetylcholine acts via the muscarinic receptor M3. Serotonin and neurotensin increase intracellular Ca²⁺, and VIP and related peptide hormones (e.g., secretin, peptide histidine leucine, and peptide histidine methionine) increase intracellular cAMP. Guanylin acts by increasing cell cGMP.

Serotonin

Serotonin plays a critical role in modulating intestinal motility, sensation, and secretion and is responsible for the diarrhea associated with carcinoid tumors. About 95% of the body’s serotonin is produced by enterochromaffin cells, and the remainder is produced by serotoninergic neurons of the myenteric plexus. Sensory receptors on enterochromaffin cells are activated by mechanical stimuli, acidity, invading pathogens, and dietary contents; for example, SGLT-like protein activates enterochromaffin cells, which serve as a glucose sensor to secrete serotonin (see Fig. 99-9).⁹⁹ Although not completely elucidated, this signaling involves a complex sequence of autocrine and paracrine actions: ATP is released and converted extracellularly to ADP, which in turn activates a purinergic (P2Y) receptor-mediated calcium signaling cascade in the enterochromaffin cell to release 5-HT.⁹⁹ 5-HT then acts in a paracrine manner to stimulate epithelial cells, intrinsic primary afferent neurons (IPANs), and extrinsic primary afferent neurons (EPANs). Specific 5-HT receptor subtypes on different IPANs modulate the secretory reflex. Thus, 5-HT_1PR on submucosal IPANs and amplifying presynaptic 5-HT₄ receptors cause the release of acetylcholine and calcitonin gene-related peptide, which stimulate peristaltic and secretory reflexes. In contrast, 5-HT₃Rs on myentric IPANs trigger the release of acetylcholine to stimulate giant migrating contractions.⁹⁹^–¹⁰¹ Serotonin stimulation of EPANs results in CNS-mediated responses of nausea and discomfort.

The major mechanism of serotonin inactivation is by a serotonin reuptake transporter (SERT) on enterocytes and neurons. Interestingly, SERT may be decreased in patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and ulcerative colitis and might account for their increased colonic motility and diarrhea. There has been a concerted effort to clinically alter intestinal function by pharmacologic manipulation of specific serotonin receptors. Thus, 5-HT₃ receptor antagonists, such as alosetron, are used to treat IBS-D, and tegaserod, a partial 5-HT₄ agonist, can alleviate constipation associated with IBS. Unfortunately, significant side effects have limited their clinical use (see Chapter 118).⁹³^,¹⁰¹^,¹⁰²

Adenosine and related purine nucleotides play unique and complex roles in modulating secretion in vivo. As described earlier, they can stimulate secretion directly as it occurs in response to polymorphonuclear leukocyte infiltration of the mucosa or indirectly via release of 5-HT.⁹⁹ Adenosine acting via P1 purinoreceptors, however, has been shown to attenuate secretion evoked by mechanical stimuli. Activation or inhibition of different populations of channels can underlie these seemingly opposite effects.

Guanylin, Nitric Oxide, and Reactive Oxygen Metabolites

The search for an endogenous activator of the E. coli heat-stable enterotoxin receptor led to the discovery of guanylin and uroguanylin, another example of molecular mimicry. These small peptides, synthesized in goblet and columnar cells activate membrane guanylate cyclase to increase intracellular cGMP and elicit fluid secretion. The guanylin family of peptides, in conjunction with the atrial natriuretic peptides, calibrate the intestinal-renal acid-base response axis. In contrast, nitric oxide, a neuroimmune regulator, stimulates soluble guanylate cyclase to increase cGMP.¹⁰³ This enzyme is far more prevalent in the subepithelium of the small intestine, but it is expressed in colonic epithelia. Reactive oxygen metabolites, including oxygen free radicals and hydrogen peroxide, generally are produced by immune cells, but they can be released by epithelial cells under certain conditions. They stimulate fluid secretion (see the discussion of immunologic regulation). Additional agonists stimulate secretion (see Table 99-2). Interestingly, most of these agents also affect intestinal motility.

Microbial Pathogens

Microbial pathogens including bacteria, viruses, and fungi can alter electrolyte transport, increase intestinal permeability, and trigger inflammation to elicit diarrhea. They do this by a variety of mechanisms, including attaching to epithelial cells to insert their own products and alter host cell machinery, and by elaborating enterotoxins, which may be cytotoxic or can capture cell-signaling mechanisms to elicit secretion or disrupt tight junctions (see Table 99-3; see also Chapter 107).¹⁰⁴^–¹⁰⁸ A few examples are provided here.

Shigella causes dysentery by release of Shiga cytotoxins, which enter the epithelial cell, inhibit protein synthesis, impair absorption, and damage the mucosa. In contrast, secretory diarrheas, such as those associated with cholera and traveler’s diarrhea, result from noninvasive pathogens, which elaborate enterotoxins that capture and turn on the secretory machinery of the epithelium. The archetypal enterotoxin-mediated diarrhea is cholera. Vibrio cholerae carry a virulence cassette that produces at least three different molecules: an enterotoxin; a zonula occludens toxin (ZOT), which disrupts tight junction permeability; and a channel-like protein.¹⁰⁹ The enterotoxin causes an unregulated increase in cAMP, activates CFTR, and inhibits NHE3, thus resulting in copious fluid secretion (see Fig. 99-9 legend for details). Mice lacking CFTR do not respond to cholera toxin. Despite voluminous secretion, specific intestinal Na⁺-coupled nutrient absorptive (Na⁺-glucose, Na⁺-amino acids) pathways are unaltered by the toxin, forming the physiologic basis for ORT. Bacteria such as Salmonella species, Campylobacter jejuni, and E. coli elaborate enterotoxins similar to cholera toxin, thereby employing the cAMP machinery to elicit fluid secretion.

Strains of E. coli and Yersinia enterocolitica associated with traveler’s diarrhea elaborate small-molecular-weight, heat-stable enterotoxins, which increase cGMP to stimulate fluid secretion; signaling cascades distinct from cAMP and cGMP also activate CFTR and inhibit NHE3 (see the discussion of intracellular mediators). Vibrio parahaemolyticus elaborates a thermostable direct hemolysin (TDH) and is a major cause of gastroenteritis; its associated intestinal fluid secretion is attributed to an increase in intracellular calcium and activation of the Ca²⁺-calmodulin and protein kinase C signaling pathways.⁹³^,¹⁰⁸

Rotavirus, the major cause of infantile gastroenteritis, induces watery diarrhea. The virus predominantly infects the mature enterocyte of the villus and elaborates an enterotoxin NSP4.¹¹⁰^,¹¹¹ NSP4 can inhibit brush border membrane disaccharidases and SGLT1 activity, thereby limiting Na⁺-glucose and fluid absorption, with resultant diarrhea. NSP4 in vitro elicits Cl⁻ secretion via a calcium-phospholipase C pathway that stimulates Ca²⁺-activated Cl⁻ channels; this effects a transient secretion similar to the actions of carbachol. Unlike other secretagogues, NSP4 has no effect on crypt cell secretion, but under favorable electrochemical conditions it stimulates secretion from villus cells. Paradoxically, NSP4 also can stimulate Cl⁻ absorption from villus cells. The role of NSP4 in vivo has yet to be defined.

Many bacterial pathogens use different signaling molecules (e.g., kinases and phosphatases) to perturb the delicate balance of tight junctional proteins and cytoskeletal elements, thus disrupting intestinal permeability. Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli decrease transepithelial resistance using different signaling cascades. The EPEC strains use a fascinating arsenal to alter host cell responses: They adhere to intestinal cells and in the process recruit a complex network of host cytoskeletal elements. Infectious bacteria use a variety of mechanisms, classified as types I to IV, to infect host cells. EPEC strains use a type III secretion apparatus to insert effector molecules into the host cell and use these molecules to co-opt the cell machinery to cause changes in the actin-myosin network, alter tight-junction proteins, and modulate ion transport processes to decrease absorption and increase fluid accumulation.¹⁰⁵^,¹⁰⁷ The anaerobic bacterium Clostridium difficile, which causes antibiotic-associated pseudomembranous colitis, and Clostridium perfringens, which is associated with food-borne illnesses, alter intestinal permeability by using two distinct processes: The C. difficile toxins A and B interact with the Rho family of cellular proteins to disrupt the perijunctional actin-myosin ring, whereas the tight-junction claudin proteins serve as receptors for C. perfringens enterotoxin (CPE), and binding results in a disruption of tight-junctional fibrils. Other bacteria, including Bacteroides fragilis and V. cholerae, elaborate proteases that attack junctional proteins, including occludins, claudins, and cadherin, to disrupt the integrity of tight junctions.⁹³^,¹⁰⁸

Many bacterial strains, including EPEC, EHEC, ETEC, Salmonella, and Shigella, trigger a highly specialized cascade to stimulate ion secretion. These pathogens induce the expression of receptors for the peptide neurohormone galanin, whereas uninfected cells do not possess galanin receptors. Galanin in turn activates Cl⁻ secretion via Ca²⁺-dependent signaling processes.¹¹²^–¹¹⁴ ¹¹⁷ The ever-expanding spectrum of toxin-induced mechanisms underscores the importance of delineating the intrinsic regulatory processes and the molecular pathophysiology of infectious diarrhea.

Bile Acids

An increase in colonic bile acids secondary to ileal malabsorption or oral supplementation can cause diarrhea (see Chapters 15 and 64).¹³^,¹⁴ Only 7α dihydroxy bile acids such as chenodeoxycholic acid (3α; 7α), but not 7β, are associated with diarrhea. At high concentrations, bile acids act as a detergent and increase intestinal permeability. At more physiologic concentrations, bile salts indirectly increase cAMP and activate mast cells and, more importantly, stimulate epithelial cell Cl⁻ secretion via the Ca²⁺ and PKCδ cascade.¹¹⁵^,¹¹⁶ The ability of bile salts to stimulate Cl⁻ secretion occurs only in the adult and is absent in the neonatal animal.

Long-chain fatty acids are seen in increased concentration in the colonic lumen in conditions such as sprue, when long-chain triglycerides are digested by lipase but the fatty acids are malabsorbed within the small intestine (see Chapters 101 and 104). Hydroxylated fatty acids are more potent secretagogues than the corresponding long-chain fatty acids and arise from colonic bacterial metabolism; ricinoleic acid is the long-chain fatty acid that is derived from oral castor oil, which is a nontoxic oil before it is hydroxylated. Specific fatty acid transporters have been identified in the intestine, and their mechanisms of action in electrolyte secretion are similar to those of bile acids.¹¹⁸^,¹¹⁹

INTRACELLULAR MEDIATORS

The barrage of extracellular stimuli need to be translated into an intracellular language so the cell can regulate its transport machinery. The second messenger cascades of the cell include the cyclic nucleotides cAMP and cGMP, intracellular Ca²⁺, and the inositol phosphate-diacyl glycerol and tyrosine kinases. These messenger systems are common to several organ systems, and many cell-specific and tissue-specific structural and functional nuances contribute to the net biological response. More detailed descriptions of second messenger systems can be found elsewhere, but an overview is provided here.¹²⁰

Epithelial cells require rapid response cascades for turning on and turning off ion transport systems. For example, cyclases are poised to synthesize cyclic nucleotides, and phosphodiesterases are set to degrade them. The net biologic response is governed by the relative contributions of the accentuating and attenuating processes. In addition to the burgeoning increase in new signaling molecules awaiting definition of their roles, there are other variables compounding the resulting net biologic response.

The molecules at almost every step in the signal transduction cascade, from the activating hormone to the receptors, cyclases, kinases, and phosphatases, and, finally, the transporters themselves, exist as multiple isoforms and variants (see Fig. 99-7). These isoforms exhibit differences in species, tissue, cell type, and subcellular distribution and are subject to regulation during their development as well as in response to routine physiologic demands.

The cannonical cascade of stimulus → second messenger → kinase → response is an oversimplification; extensive crosstalk exists between different signaling pathways. Other post-translational modifications such as glycosylation, myristoylation, nitration, and sumoylation are increasingly recognized as important modulators. Within a signaling cascade there are critical feed-forward and feedback regulatory steps.

Thus, protein kinases catalyze the transfer of the terminal phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine of a target protein, leading to both conformational and functional changes, such as altered affinity for substrate. They exhibit specificity in their activators and substrates, and their action is essentially irreversible in living cells. Phosphoproteins can only be dephosphorylated by protein phosphatases, which are a separate class of enzymes that are also subject to regulation. Phosphoproteins may be the transporters themselves or may be modulator proteins in the membrane or cytosol, or they may be both. Equally important is the recognition that protein phosphorylation is not synonymous with activation; the dephosphorylated protein may be the active form. In general, Ca²⁺-specific and cyclic nucleotide-specific protein kinases are serine-threonine kinases, whereas tyrosine-specific protein kinases are associated with receptors of cytokines and hormones involved in growth, such as EGF. In addition to specific serine-threonine and tyrosine phosphatases, dual-specificity kinases and phosphatases add to the complexity of crosstalk.

Compartmentalization of components of the signaling cascade via cytoskeletal runners, anchoring domains, or sequestration in vesicles as a means of regulation is especially germane to the polarized enterocyte. Localizing transporters and their signaling systems into specific subcellular domains is the norm rather than the exception. This is a dynamic and highly regulatable process. Scaffolding proteins can promote docking of various proteins, kinases, and phosphatases by protein-protein interactions—to each other, to the cytoskeleton, and/or to the membrane (see Fig. 99-7); for example, guanylate cyclase C and the intestinal protein kinase GII have cytoskeletal and membrane interacting domains that bring them in close proximity to CFTR in the brush border membrane (Fig. 99-7). Cholesterol-rich membrane domains such as lipid rafts influence membrane fluidity and anchor specific transporters and their regulators. It should come as no surprise that many anchoring domains serve as multienzyme signaling complexes. Finally, trafficking of transporters into and out of the membrane via endosomal vesicles is an effective way of rapidly altering the V_max of the transporter. For example, cAMP increases CFTR translocation to the membrane and NHE3 retrieval from the membrane, resulting in an increase in Cl⁻ secretion and decrease in Na⁺ absorption (see Fig. 99-9). Thus, the cAMP-stimulated increases in Cl⁻ secretion and decreases in Na⁺ absorption (see Fig. 99-9), respectively, result from an increase in CFTR translocation to, and NHE-3 retrieval from, the membrane.

Although it is not associated with rapid responses, neurohumoral stimulation can cause changes at the transcriptional level, leading to the synthesis of new proteins, such as aldosterone, which increases ENaC synthesis in the distal colon. Diseases such as cystic fibrosis underscore the importance of intracellular quality control in the cell machinery; thus ΔF508 CFTR, the most common mutation, is a misfolded protein and is tagged for degradation. All signal-transduction mechanisms need to be assessed with respect to their physiologic relevance in the intact intestine because reductionist models, although a necessity, do not provide the complete picture. With these caveats, some common themes have emerged.

Generally, agents that elevate intracellular cAMP, cGMP, or Ca²⁺ increase fluid secretion (see Tables 99-2 and 99-3). They can activate one or more transporters associated with electrogenic Cl⁻ secretion: apical Cl⁻ and K⁺ channels, basolateral K⁺ conductances, and NKCC1; they also inhibit the apical NHEs, NHE2 and NHE3. Conversely, fluid absorption is associated with a decrease in these messengers or with an activation of some tyrosine kinase pathways. cAMP plays an additional role in promoting trafficking of transporter-bearing vesicles (CFTR in crypts and distal colonic Na⁺ channels) to the apical membrane.⁹¹^,¹¹¹

The cAMP cascade is triggered by a hormone, such as VIP, binding to a specific member of the superfamily of heptahelical membrane-spanning receptors (7TM–VPAC1 and VPAC2) (see Fig. 99-7).¹²¹ Cyclic GMP is generated by the activation of membrane or soluble guanylate cyclases (GCs) by the natriuretic peptides guanylin and uroguanylin (see Fig. 99-7). Guanylins share their receptor GC-C with the heat-stable enterotoxins (see the earlier discussion of guanylin). Hormones and neurotransmitters such as substance P and acetylcholine activate secretion by increasing intracellular Ca²⁺ (Fig. 99-11; see figure legend for details).