I. INTRODUCTION

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The epithelial layer covering the inner surface of the mammalian colon is a typical electrolyte-transporting epithelium, which is able to move large quantities of salt and water from the mucosal side toward the blood side or vice versa. Under physiological conditions, fine tuning of salt excretion in the stool is achieved by colonic absorption of ~1.5 l of electrolyte-rich fluid per day. The basic concepts of ion transport in the colon have been elucidated some time ago (123, 264), but only during the past few years the responsible proteins have been identified. Polarized colonic epithelial cells are equipped with a number of ion channels, carriers, and pumps, located either on the luminal or basolateral membrane, allowing highly efficient transport of large amounts of salt and water. In this review, we summarize the current knowledge on the molecular nature of these transport proteins. We will further outline their regulation and interaction with additional regulatory proteins. The ion transport activity of the epithelium leads to net absorption of electrolytes under control conditions and secretory properties become apparent only after stimulation by secretagogues. Net transport is the result of well-balanced absorption and secretion.

In contrast to what has been assumed previously, recent data show that ion transport in the absorptive or secretory direction is present in both surface epithelium and crypts of Lieberkühn (336). We have just begun to understand how an individual colonocyte can cope with both absorption and secretion and how it is able to switch from absorption to secretion when stimulated by secretagogues. Ion transport is disturbed during infectious diseases causing secretory diarrhea, which can cause life-threatening dehydration by excessive loss of salt and water (174, 175). In the case of cystic fibrosis, excessive absorption leads to intestinal dehydration and obstruction presenting with meconium ileus at birth, a distal intestinal obstruction syndrome (DIOS), and chronic constipation with rectal prolapse in older cystic fibrosis (CF) patients (49). These examples demonstrate that net transport can be excessive under pathological conditions and indicate that both secretion and absorption have to be tightly regulated to maintain proper net transport.

The purpose of this review is to give a summary of the molecular aspects of electrolyte transport in the colon and describe new aspects of regulation of the participating transport proteins. Our focus is on the native tissue rather than cultured colonic epithelial cells. We do not aim to present a detailed review on molecular properties of the Na⁺-2Cl-K⁺ cotransporter or the Na⁺-K⁺-ATPase, which have been reviewed extensively elsewhere (203, 240, 509). Instead, we focus on other aspects of electrogenic and electroneutral ion transport. This includes the contributing epithelial ion channels and transporters as well as their regulation in proximal and distal colon. We also describe specific aspects of dysfunction of epithelial transport, under pathophysiological conditions during secretory diarrhea and CF. Other membrane transport processes occurring in the colonic epithelium, such as absorption of short-chain fatty acids (SCFA) and secretion of mucus are only discussed in the context of ion transport. They have been reviewed elsewhere (146, 184, 247, 428).

	II. ANATOMY AND TASKS OF THE COLONIC EPITHELIUM

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The primary nonmotor function of the human colon is absorption of ~1.3-1.8 liters electrolyte-rich fluid per day, which accounts for ~90% of the salt and water entering the proximal colon (125). Most data on colonic transport were obtained in studies on colonic tissues from rat, rabbit, mouse, and human. Despite considerable quantitative differences, the mechanisms are qualitatively very similar in the different species (123, 665). Epithelial cells coating the inside of the mammalian colon form a low-resistance epithelium in the proximal colon of only ~100 ·cm². The resistance is about two- to fourfold higher in rat distal colon, which can therefore be described as a "moderately" or "medium" resistance epithelium (100, 101, 667). The values are about twice as high in the mouse distal colon. Here the paracellular resistance is high compared with transcellular resistance (212). Colonic epithelial cells are highly conductive, with a greater density of ion channels in the luminal membrane of surface compared with crypt cells. The result of ion transport is excretion of a stool containing <5 mM Na⁺, 2 mM Cl, and 9 mM K⁺ (645). In addition to electrogenic and electroneutral absorption of NaCl, active absorption of K⁺ by luminal K⁺ pumps and absorption of SCFA produced by the intestinal flora are primary tasks of the colonic epithelium. Although net absorption of NaCl, KCl, and water are dominant housekeeping functions of the colon, secretion of NaCl and KCl also takes place and largely exceeds absorption in secretory diarrhea. In addition to the transport of NaCl and KCl, the colonic epithelium secretes HCO and mucus. Both have been demonstrated to have important protective and milieu functions.

According to morphological studies, at least three different cell types form the mammalian colonic epithelium. They comprise columnar epithelial cells, mucous, and argentaffin cells (84, 610). Columnar epithelial cells and goblet cells contribute to ~95% of all cells. In addition, enterochromaffin (enteroendocrine) cells make up another 5%. A surface epithelial layer is differentiated from colonic crypts. The columnar epithelial cells can be subdivided according to their degree of differentiation, which is based on their proliferative activity, expression of differentiation markers, and functional properties (266, 268, 274, 498, 506). Thus base crypt epithelial cells show the highest proliferative activity, demonstrate limited expression of differentiation markers, and have a high Cl secretory activity. In contrast, surface epithelial cells have a lower tendency to proliferate, show expression of differentiation markers and certain lectins, and have primarily absorptive function (266, 268, 337). The cells become increasingly differentiated the further they are located away from the crypt base and the closer they are to the surface. Thus highly proliferative and fairly undifferentiated epithelial cells in the crypt base form a constant source for replacement of the surface cells. These replacing cells differentiate while traveling along the crypts toward the surface (46, 157, 158) (Fig. 12).

It is still a matter of debate whether crypts and surface epithelium represent two specialized compartments with distinct functions in either absorption (surface epithelium) or secretion (crypts) of electrolytes (175, 659). It has been suggested that secretion occurs to clear the crypts from mucus, which is secreted from goblet as well as columnar epithelial cells (247). However, mucus secretion also takes place in the surface epithelium. Therefore, this and further studies do not support the idea of exclusive secretion in the crypts. There is now clear evidence that electrolyte secretion is located in both surface epithelium and crypts (336). This comes from studies using vibrating electrodes (294, 336) as well as patch-clamp studies in which Cl channels could be demonstrated in both crypts and the surface (144). Moreover, CFTR Cl channels show a gradient of expression along the crypt/villus axis (606). Functional analysis of cAMP-activated Cl conductance and in situ hybridization suggest highest expression of CFTR in crypt cells, and studies in cultured colonic epithelial cells indicate a higher mRNA expression in undifferentiated cells (220, 577, 585). Interestingly, it has been shown by in situ hybridization that expression switches from cystic fibrosis transmembrane conductance regulator (CFTR) to MDR1 as the cells migrate across the crypt/villus boundary. Thus coordinated regulation of expression of these two genes has been assumed (607). However, expression of mRNA and CFTR Cl currents may not necessarily be tightly correlated (29). Nevertheless, CFTR Cl channels expressed in surface epithelial cells may be required for absorption rather than secretion of NaCl, since they are colocalized together with epithelial Na⁺ channels (ENaC). This and the fact that CFTR is likely to serve as a regulator of ENaC are outlined later in this review (341) (Figs. 3 and 4).

The localization of absorption within the colonic epithelium is even more controversial. As outlined below, absorption can be electrogenic via the ENaC or is electroneutral via parallel Na⁺/H⁺ and Cl/HCO exchange. According to the work of some groups, the high osmotic pressure gradient that is necessary to absorb water from the intestinal lumen and to generate the mammalian feces can only be created by a trapping mechanism for ions located in the crypts rather than the surface epithelium (434, 435, 457, 571). These interesting results are based on fluorescence shifts using fluorescent dextrans. At any rate, electroneutral NaCl absorption has been shown to take place in both crypts and surface epithelium (571) (Fig. 1).

View larger version (39K): [in this window] [in a new window]   Fig. 1. Models for electrolyte transport in proximal and distal colonic epithelium and expression of different ion transporters along the crypt axis. Electroneutral NaCl absorption (parallel Na+/H+ and Cl/HCO exchange) dominates in the surface epithelium and is also present in the crypts. Electrogenic Na+ absorption via the epithelial Na+ channel (ENaC) takes place in the surface epithelium and upper crypts of the distal colon. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed throughout the colonic epithelium and dominates in the crypts.

In contrast, electrogenic absorption via ENaC is confined to the surface epithelium and upper part of the crypt, as determined by voltage scanning and whole cell patch-clamp experiments (220, 337). Expression of ,,-ENaC subunits is limited to the surface and upper crypt, as verified by immunolabeling of ENaC (202). Accordingly, steroid-dependent regulation of ENaC expression was observed in the surface epithelium but not the crypts (202). The conclusion from the various studies is that bulk absorption occurs via electroneutral NaCl transport and takes place in both crypts and surface epithelium of proximal and distal colon. Evidence for electroneutral absorption in the crypts is more indirect, due to a lack of electrogenic Na⁺ absorption (39). The transport in rat and mouse colon is dominated by electroneutral absorption. Electrogenic absorption via luminal ENaC is confined to the surface epithelium of the distal colon and shows a larger contribution in rabbit, human, and guinea pig than in rat and mouse (337, 397, 465). In general, less detailed information is currently available for human and mouse colon than that of rat (50, 230, 513) (Fig. 1).

	III. ABSORPTIVE FUNCTION OF THE COLONIC EPITHELIUM

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Bulk transport of NaCl in the colonic epithelium is due to electroneutral absorption by luminal Na⁺/H⁺ and Cl/HCO exchange. The remaining absorption is electrogenic and is due to absorption via luminal ENaC and transcellular/paracellular absorption of Cl. The contribution of paracellular Cl absorption might be limited by the paracellular shunt resistance, which is roughly 20 times larger than the transepithelial resistance (212). However, the presence of a large lumen-negative transepithelial voltage, particularly in glucocorticoid-treated animals, and the fact that the paracellular shunt is not strictly ion selective, would allow for paracellular movement of Cl (5, 666, 667). Moreover, tight junctions consist of a complex array of proteins such as occludin, claudin, and paracellin and are probably actively regulated (5, 168, 569, 678). Thus it has been demonstrated that the tight junction permeability of the rat ileum is increased by cAMP (38).

There is a clear segmental heterogeneity with respect to Na⁺ absorption present in ascending (proximal) and descending (distal) colon in human and other species. In the ascending colon, Na⁺ transport is primarily mediated by an electroneutral process, while Na⁺ transport in the descending colon is dominated by electrogenic absorption via amiloride-sensitive Na⁺ channels under the influence of aldosterone (101, 367, 518, 679) (Fig. 1). In the absence of steroids, electroneutral absorption is the predominant transport process in both rat proximal and distal colon (39, 186, 187, 192). It should be mentioned, however, that significant species differences exist regarding the contribution of electroneutral and electrogenic Na⁺ absorption. For example, while electrogenic absorption dominates the rabbit distal colon, the rat colon is dominated by electroneutral absorption (39, 465). Limited information is available for human and mouse colon. For electroneutral absorption of NaCl, the presence of parallel Na⁺/H⁺ and Cl/HCO exchangers in luminal brush-border membranes of colonic epithelial cells is required (Fig. 2). It is driven by the action of the basolateral Na⁺-K⁺-ATPase and probably requires 1 mol ATP being hydrolyzed per 3 mol NaCl absorbed (307). The transport of Na⁺ and Cl is coupled via changes in intracellular pH and Cl (204, 480, 485). It is regulated by Na⁺ depletion or steroids (27, 186, 282, 671). The properties of these transport proteins are discussed in more detail below.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Cellular model for electroneutral absorption of NaCl in the mammalian colon. Na+ is taken up from the luminal side of the epithelium by Na+/H+ exchangers type 2 and 3 (NHE2 and NHE3). The Na+/H+ exchange regulatory factor (NHERF) interacts with NHE3 and is required for cAMP-dependent inhibition of NHE3. Luminal NHE activity is paralleled by luminal Cl/OH and Cl/HCO exchange, due to the proteins DRA (downregulated in adenoma) and anion exchanger type 1 (AE1). Basolateral anion exchange occurs via anion exchangers type 1 and 2 (AE1 and AE2). Although clear evidence was found for expression of the basolateral KCl cotransporter KCC1, a contribution of Cl channels to electroneutral absorption remains to be confirmed by additional studies. The basolateral Na+-K+-ATPase generates the driving force for the luminal Na+ uptake by lowering intracellular Na+ concentration. Basolateral cAMP and Ca2+-activated K+ channels form the recycling pathway for K+.

Expression of three types of the Na⁺/H⁺ exchanger (NHE) has been detected so far in the colonic epithelium. The abundant type 1 NHE is expressed in the basolateral membrane and does not seem to be affected by Na⁺ depletion. NHE2 and NHE3 are both expressed on the luminal side of colonic epithelial cells, with a larger contribution of NHE3 to Na⁺ absorption under control conditions (282). Regulation of NHE2 and NHE3 differ in proximal and distal colon, in as much as expression of both types is upregulated by Na⁺ depletion in the proximal colon, but is attenuated in the distal colon (282, 480). In addition to NHE2 and NHE3, a third and novel type of Cl-dependent NHE has been identified in apical membranes of rat crypt cells (485). This transporter is upregulated by Na⁺ depletion and increases in plasma aldosterone (487). According to ²²Na⁺ uptake studies, the transporter is functionally coupled to a Cl channel rather than a Cl/anion exchange mechanism, and some data suggest that this Cl channel is identical to CFTR (486). Moreover, Na⁺/H⁺ exchange is also detected on basolateral membranes and is due to the housekeeping function of the type 1 NHE (257, 282, 463).

Na⁺/H⁺ exchange occurs in both surface and crypt epithelium, is tightly coupled to Cl/HCO exchange, and might be controlled by CFTR Cl channels (480, 485, 486). Although little is known about the impact of CFTR on cAMP-dependent regulation of the Na⁺/H⁺ exchange in the colon, clear evidence has been found in the small intestine. In the normal (non-CF) human intestinal epithelium, an increase in intracellular cAMP inhibits electroneutral reabsorption of NaCl (43). In contrast, cAMP-dependent activation is not observed in the small intestine of CF patients carrying a defective CFTR. In the CF jejunum, an increase in intracellular cAMP even further activates absorption of Na⁺ (35, 446). Similar to the results obtained in the human mucosa, stimulation of the intestine of wild-type mice by cAMP inhibits electroneutral absorption of NaCl. This is not observed in the intestine of CFTR (/) knockout mice (98). Therefore, it is likely that CFTR also regulates electroneutral absorption of NaCl in the colon. In renal epithelial cells, a Na⁺/H⁺ exchanger regulatory factor (NHERF) has been identified that is required for cAMP-mediated inhibition of Na⁺ absorption by luminal NHE3 and basolateral Na⁺/HCO cotransporter (34, 655, 681). Transcripts for NHERF and the homologous protein E3KARP (NHE3 kinase A regulatory protein), were also identified in small intestinal epithelial cells but not in cultured colonic carcinoma cells (681). Interestingly, regulation of NHE3 by CFTR also requires the presence of NHERF, as demonstrated in a heterologous expression system and in mouse pancreatic ducts (2). In summary, the contribution of CFTR and NHERF to the regulation of Na⁺/H⁺ exchange in the colonic epithelium is likely, yet still needs to be demonstrated. Detailed information regarding the function of NHERF in epithelial cells is given in two recent review articles (557, 654).

Functional studies have shown the presence of at least two different types of Cl exchange mechanisms in apical membranes of colonic epithelial cells. In addition, a third type of Cl/HCO exchange is expressed in the basolateral membrane (484, 541, 594). The identified luminal Cl/HCO and Cl/OH exchange are represented by the anion exchanger type 1 (AE1) and a protein called DRA, which stands for downregulated in colonic adenomas (484, 541). DRA has been demonstrated to function as a transporter for SO, as well as an anion exchanger (3, 546). Interestingly, DRA is upregulated in mice lacking the NHE3 but is otherwise not regulated by Na⁺ depletion, whereas expression of AE1 is inhibited by aldosterone (269, 417, 484, 541). Mutations in human DRA are responsible for congenital chloride diarrhea and the most common form of syndromic deafness, described as the Pendred syndrome (269, 429, 546). Interestingly, in the airways, expression of DRA depends largely on the expression of the CFTR, a finding which could also apply to the colon (661). The Cl/HCO exchange that is present in basolateral membranes of colonic epithelial cells is due to expression of the isoforms AE1 and AE2 (Fig. 2) (39, 482, 594). Like NHE3, Cl/HCO exchange is also likely to be controlled by CFTR in the colonic epithelium. Regulation of Cl/HCO exchange by CFTR has been demonstrated for other parts of the gastrointestinal tract, such as submandibular and pancreatic ducts. It is also observed after heterologous expression in NIH3T3 or HEK 293 cells (361, 362, 388). Taken together, the current studies indicate the presence of several types of Na⁺/H⁺ exchanger, along with three different anion exchangers. The results further suggest a regulation of both Na⁺/H⁺ as well as Cl/HCO exchangers by CFTR, which therefore has an impact on electroneutral absorption of NaCl and regulation of cellular and mucosal pH in the colonic epithelium and other sections of the gastrointestinal tract (214, 466, 653).

In addition to electroneutral absorption by parallel exchange of Na⁺/H⁺ and Cl/HCO, epithelial cells of the distal colon possess a mechanism for electrogenic uptake of Na⁺. The ENaC is responsible for electrogenic absorption and is located in the luminal membrane of colonic epithelial cells. ENaC is potently inhibited by amiloride and related diuretic compounds (18, 31, 74, 75, 202, 497). In addition to ENaC, other types of cation channels have been detected in the cecum (552, 553). However, the quantitative impact of these channels on electrolyte absorption remains to be demonstrated. The basic concept of electrogenic Na⁺ absorption has been elaborated many years ago (123, 330). Due to the electrochemical gradient for Na⁺ and the negative cell membrane voltage, there is a large driving force for luminal Na⁺ uptake via ENaC. Absorption of Na⁺ is accompanied by the counterion Cl, which is taken up by Cl channels, localized in the apical membrane of absorptive epithelial cells, and is also likely to occur via the paracellular shunt, as outlined in section IIIA (220, 347, 397). CFTR is the predominant luminal Cl channel in the colonic epithelium (396). Whole cell patch-clamp experiments and in situ hybridization suggested coexpression of CFTR and ENaC in surface and midcrypt epithelial cells of the rat colon (157, 220, 606). Due to the large driving force for Na⁺ uptake in these cells and the depolarization of the luminal membrane voltage, we speculate that CFTR Cl channels may also serve as an absorptive pathway for Cl in these cells (Figs. 1 and 3). Thus the situation could be somewhat similar to that of the sweat duct (493). However, due to the inhomogeneous architecture of the native colonic epithelium and the lack of Na⁺ transport in cultured colonic cells, it has not been possible to demonstrate Cl absorption by CFTR in the colon. In contrast, crypt base cells express large amounts of CFTR but no ENaC, and therefore, CFTR has clearly a secretory function in this part of the colonic epithelium (220, 606). Na⁺ that have been taken up into the cell are pumped out again on the basolateral side of the epithelium by the Na⁺-K⁺-ATPase. Cl that have entered the cytosol via apical Cl channels leave the cell via basolateral Cl channels or Cl/HCO exchangers (220, 473, 657). Water can move via various pathways, including the paracellular shunt and the transcellular flux through aquaporin water channels located in both luminal and basolateral membranes. However, a major role for aquaporins in the colon has not yet been established (640). In addition, water transport may potentially occur via substrate transporters (384, 415, 416, 682). The different membrane proteins participating in absorption of NaCl have to be regulated in parallel. Basolateral outward transport of Na⁺ by Na⁺-K⁺-ATPases must keep up with the apical Na⁺ entry via Na⁺ channels (222, 225). The putative Na⁺ feedback mechanism and other regulatory functions are discussed in the following sections.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Cellular model for electrogenic absorption of NaCl in the mammalian colon. Na+ is taken up from the luminal side of the epithelium by epithelial Na+ channels (ENaC), formed by 2/1/1 subunits. The basolateral Na+-K+-ATPase generates the driving force for the luminal Na+ uptake by lowering intracellular Na+ concentration. Basolateral cAMP and Ca2+-activated K+ channels form the recycling pathway for K+. Na+ uptake is generating a large lumen-negative transepithelial voltage that facilitates Cl absorption through luminal CFTR and/or other luminal Cl channels and eventually a Cl conductive paracellular shunt pathway. The CFTR in these absorptive epithelial cells leads to inhibition of ENaC and a decrease in NaCl absorption. Basolateral KCl cotransporter (KCC1), Cl channels, and anion exchangers type 1 or 2 (AE1/2) may transport Cl to the blood side of the epithelium. K+ secretion to the luminal side of the epithelium is driven by electrogenic uptake of Na+.

ENaC are expressed on apical membranes of absorptive colonic epithelial cells. These channels are highly selective for Na⁺ over K⁺ and have a rather small single-channel conductance of ~4 pS and a linear current-voltage relationship (249). So far, most studies have been done on other tissues, and single-channel analysis of ENaC on the colonic epithelium is not yet available. Na⁺ uptake by ENaC into colonocytes is the rate-limiting step during electrogenic absorption of Na⁺ (201). The amiloride-sensitive ENaC has been cloned initially from rat colon. It consists of three different subunits (-, -, and -ENaC) (74, 75). Probably four subunits (2-, 1-, 1) coassemble to form a functional Na⁺ channel (73, 179, 202). However, other studies claim that ENaC is composed of 9 subunits or even 17 transmembrane -helices (164, 575). ENaC belongs to a large family of related proteins, which is discussed in several excellent review articles (4, 18, 73, 202, 450). The large extracellular domains of the ENaC subunits contain cysteine-rich boxes, implying a receptor-like structure (255, 623). Indeed, a serine protease (mouse channel activating protease; mCAP) was recently found to be coexpressed with ENaC in absorptive epithelial cells. It may enhance channel activity via interaction with the extracellular loop of the channel (93, 623). ENaC subunits, which traverse the lipid membrane only twice, contain proline-rich segments in the intracellular COOH terminus of each subunit (581). These segments are essential for the interaction with the ubiquitin ligase Nedd4, which leads to ubiquitination and endocytosis of the channel protein (581). Mutations in these proline-rich segments of different subunits lead to a salt-sensitive form of hypertension due to excessive absorption of Na⁺ in the kidney collecting duct in Liddle's disease (505).

D.  Regulation of Na⁺ Absorption

1.  Feedback regulation

Recent studies demonstrate the importance of the COOH-terminal PY motifs present in all three ENaC subunits, for the so-called "feedback inhibition" of ENaC. Feedback inhibition describes the phenomenon that Na⁺ has a negative impact on the activity of Na⁺ channels (202, 614). Changes in the intracellular Na⁺ concentration during NaCl absorption have been suggested to downregulate ENaC conductance. This mechanism causes a negative feedback loop, which controls luminal entry of Na⁺ and thus absorption of NaCl (148, 202, 331). Na⁺ feedback was demonstrated in turtle and rabbit colon some years ago (326), but until recently, the mechanisms of the inhibition of electrogenic Na⁺ absorption remained unclear (614-616). Interestingly, mutations causing Liddle's disease reduce Na⁺-dependent downregulation of ENaCs in Xenopus oocytes (317). Recent studies examined the feedback regulation in more detail in mouse mandibular duct cells and demonstrated suppression of ENaC whole cell currents by enhanced intracellular Na⁺ concentration (149). This inhibitory pathway requires sensing of the intracellular Na⁺ concentration via an intracellular Na⁺-sensitive structure and also includes activation of certain subtypes of GTP binding proteins (68, 147, 148). Activation of these G proteins leads to binding of Nedd4 to ENaC with subsequent ubiquitination and a possible endocytotic retrieval of Na⁺ channels (147).

Interestingly, a very similar mechanism for Na⁺ feedback regulation was identified recently for Na⁺-dependent regulation of the NHE in mouse mandibular duct cells. However, this process does not seem to require the ubiquitin protein ligase Nedd4 (288). Along the same lines, an increase in intracellular Cl concentration was found to inhibit ENaC via activation of a different subtype of G protein (331). Similar to the Na⁺ feedback, Cl entry into colonocytes may trigger downregulation of Na⁺ channel activity and eventually endocytosis of ENaC. How luminal CFTR Cl channels and eventually other Cl channels may contribute to this process is outlined in section IIID4.

Electroneutral absorption is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase-coupled receptors, and protein kinases, which are summarized in a recent review (151). Activation of protein kinase C (PKC), Ca²⁺/calmodulin-dependent kinase, and increases in intracellular cAMP inhibit NHE3, whereas stimulation of ₁- or ₂-receptors activates NHE3 (246, 271, 377, 504, 609). Increases of intracellular cAMP inhibit the activity of NHE3, by a mechanism involving additional scaffolding proteins such as NHERF and the cytoskeleton binding protein ezrin (655, 681). Electrogenic reabsorption of Na⁺ and water occurs at a rather steady rate and does not seem to be affected by intestinal peptide hormones or autonomic nerve activity. In fact, early studies indicated that the rat colonic mucosa is set to almost maximal absorption and that the impact of regulatory mechanisms is typically to reduce absorption and to induce electrolyte secretion (7). Thus activation of electrolyte absorption in the intestinal epithelium by endogenous hormones is of limited importance (66, 252). Hormones such as norepinephrine (acting on -receptors), somatostatin, and peptide neurotransmitters such as peptide YY and neuropeptide Y (NPY) can increase electrolyte absorption by direct action on enterocytes (66, 140, 376, 604). However, an increase in net absorption is typically through inhibition of secretion (66, 140, 604).

Other forms of immediate regulation of ENaC have been described for some epithelial tissues. They include inhibitory effects of Ca²⁺ and PKC, activation of ENaC by the cAMP-dependent pathway, and the impact of actin filaments on channel activity (13, 18, 32, 76, 202, 291, 334, 562). Moreover, inhibition of epithelial Na⁺ absorption by intracellular cGMP has been recently reported (620). However, rat ENaC expressed in heterologous expression systems such as Xenopus oocytes is not activated by protein kinase A (PKA) (13, 64, 399, 561, 562). There is some evidence for phosphorylation of - and -subunits of ENaC via PKA and acute activation of ENaC in the kidney by increases in intracellular cAMP (562). In contrast, these effects were not detected in the rat colon (62, 63). In a patch-clamp study with isolated rat colonic crypt cells, ENaC was inhibited in parallel with activation of CFTR by an increase in cytosolic cAMP (157). Similar observations were made in Ussing chamber experiments on human colonic and rectal mucosa, where activation of Cl secretion by increases in intracellular cAMP was paralleled by inhibition of amiloride-sensitive Na⁺ transport (397). Moreover, when rat ENaC was expressed in Madin-Darby canine kidney cells or NHI3T3 fibroblasts, amiloride-sensitive Na⁺ currents were enhanced by PKA in the absence of CFTR but were inhibited when CFTR was present (586, 588). Surprisingly, ENaC isolated from Xenopus kidney and guinea pig colon were shown to be activated by cAMP (372, 528). According to some studies, the effects of PKA are largely controlled by the presence of actin filaments (292), but so far it is not clear how important actin is for the regulation of Na⁺ channels. Taken together, the present results and the lack of conserved PKA phosphorylation sites in the ,,-ENaC subunits in different species suggest additional, as yet unknown, proteins that are phosphorylated by PKA and that may activate ENaC in the absence of CFTR (528). Regulation of ENaC by PKA could be tissue specific, as has reported for PKC (81).

Stimulation of purinergic receptors by extracellular nucleotides such as ATP or UTP is emerging as another mechanism for the acute regulation of colonic Na⁺ transport. Purinergic receptors are located on both luminal and basolateral membranes of rat colonic epithelial cells (323, 363). It has been shown that stimulation of these P_2Y2 receptors induces an increase in intracellular Ca²⁺ and a transient activation of KCl secretion (323). Stimulation of purinergic receptors has also been shown to inhibit electrogenic absorption of Na⁺ in airway, kidney, and thyroid epithelial cells (54, 112, 133, 215, 287, 334, 402, 490). The mechanism of nucleotide-mediated inhibition of Na⁺ absorption is not yet defined, but different models have been suggested, such as an increase in intracellular Ca²⁺ (133, 287, 402), activation of PKC (334), and a possible contribution of GTP binding proteins (270, 440). Whether purinergic inhibition of Na⁺ absorption also takes place in the colonic epithelium of rat and human remains to be determined. In unpublished experiments performed in our laboratory, we found no evidence for functional expression of purinergic receptors on either luminal or basolateral sides of the native human and rabbit colonic epithelium. In contrast, in the mouse colon, both ATP and UTP elicited large Cl currents and inhibit ENaC. The results from these Ussing chamber experiments remain to be confirmed by patch-clamp analysis of isolated human colonic epithelial cells.

ENaC, NHE3 and CFTR are coexpressed in colonic epithelial cells (157, 220, 606). This has been outlined in section IIIB. Evidence has grown over the past few years that CFTR regulates both electroneutral as well as electrogenic absorption of electrolytes in the intestinal epithelium (98, 229, 397). The impact of CFTR on Na⁺ absorption is probably twofold: in human airways and colon expressing wild-type CFTR, amiloride-sensitive Na⁺ absorption is significantly reduced compared with that of CF patients, even in the absence of cAMP, i.e., without activation of CFTR. After stimulation of wild-type CFTR by cAMP, amiloride-sensitive Na⁺ absorption is further inhibited. Thus the presence of wild-type CFTR seems to inhibit ENaC even in the absence of secretagogues. Similar has been observed in Xenopus oocytes coexpressing CFTR and ENaC (301, 348, 395, 397, 404). Enhanced amiloride-sensitive short-circuit currents have been detected in CF airways and intestine (51, 52, 229, 328, 366, 397, 449, 586). In addition to these Ussing chamber studies on the native human epithelium, inhibition of amiloride-sensitive Na⁺ conductance has been observed in patch-clamp studies on freshly isolated rat colonic epithelial cells (157). Furthermore, numerous in vitro studies and experiments in cultured cells have demonstrated interaction of CFTR and ENaC (366, 399, 586). Further insight into the regulation of Na⁺ transport was gained by the development of CFTR (/) knockout mice. CFTR has been shown to inhibit electroneutral absorption of NaCl in the mouse small intestine and pancreatic duct (2, 43, 98). CFTR knockout mice also show enhanced amiloride-sensitive short-circuit currents in the colonic epithelium compared with normal mice. These results correspond to measurements on the human CF colon, where enhanced Na⁺ absorption and lack of CFTR-dependent inhibition of ENaC was found (397). Thus CFTR may have a dual function in the mammalian colon. In the lower crypts, it is a cAMP-regulated Cl channel, essential for Cl secretion. In the upper crypt and particularly the surface epithelium, it may regulate other transport proteins such as ENaC and NHE3 (Fig. 4). In the mouse pancreatic duct, NHE3 is inhibited by an increase in intracellular cAMP even in the absence of CFTR. However, inhibition by cAMP is augmented when CFTR is present (2). Inhibition of ENaC by CFTR and current efforts in identifying the mechanism for this interaction are summarized in recent reviews (341, 352, 355, 542).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Impact of the CFTR on Na+ absorption and HCO secretion in the colon. Activation of CFTR inhibits both electroneutral Na+ absorption by the Na+/H+ exchanger NHE3 and electrogenic Na+ absorption by the epithelial Na+ channel ENaC. CFTR interacts with NHE3 via the NHE regulatory factor NHERF. HCO secretion in the colon probably occurs via electrogenic HCO secretion and the Cl/HCO exchanger. CFTR controls HCO secretion by 1) acting as a HCO-permeable Cl channel, 2) allowing recycling of Cl which has been taken up by the luminal Cl/HCO exchanger, and 3) directly activating the luminal Cl/HCO exchanger.

Interestingly, CFTR does not inhibit ENaC in the sweat duct epithelium. Here, CFTR is actually required for upregulation of ENaC and cAMP-dependent activation of Na⁺ absorption (473, 494). Correspondingly, not enhanced but rather a decreased Na⁺ conductance was detected in CF sweat ducts. The reason for the different regulation is currently unexplained but could be due to expression of additional, as yet unidentified proteins participating in the functional interaction (352). It may also reflect different morphological and functional properties of airways/colon and sweat duct. Colonic and respiratory epithelia consist of different types of epithelial cells, which show rather poor electrical coupling via gap junctions, at least in the colon (293). Therefore, individual cells seem to operate as single functional units. This is in contrast to the sweat duct epithelium, which operates as a syncytium of a single type of epithelial cell. These cells are intimately coupled via gap junctions and are devoted exclusively to absorption of electrolytes (220, 494). Moreover, the sweat duct is formed by a rather tight epithelium with a high paracellular resistance. Therefore, and in contrast to the colonic mucosa, electrolyte transport occurs only transcellularly (494). In the intestinal epithelium and probably in the airways, the situation is different since 1) epithelial cells work as individual units, and 2) they transport electrolytes in both secretory and absorptive directions. A change of vectorial transport could be achieved by inverse regulation of luminal CFTR and ENaC. In absorptive cells, coexpressing both CFTR and ENaC, activation of CFTR Cl channels would allow entry of Cl into the cell, which might shut off ENaC channels (64, 149). Thus inhibition of ENaC and NHE3 by CFTR provides a mechanism by which colonic epithelial cells limit absorption of NaCl, avoid cell swelling, and eventually switch from absorption under resting conditions to secretion when exposed to secretagogues. In summary, CFTR inhibits both electroneutral absorption of NaCl as well as electrogenic absorption of Na⁺. It therefore contributes to redirection of epithelial ion transport in the colonic mucosa by switching epithelial cells from absorption toward secretion in the presence of secretagogues.

Recently, another regulatory protein, the epithelial channel activating protease 1 (CAP1), has been identified that upregulates epithelial Na⁺ currents (623, 634). CAP1 is homologous to human prostasin and is coexpressed with ENaC in epithelial tissues, such as the cortical collecting duct of the kidney and in the colon. This protein is a serine protease that is secreted to the luminal side of the epithelium, where it interacts with the large extracellular domain of ENaC. Although clear evidence exists for CAP1 acting as a serine protease, no evidence was found for cleavage of the extracellular loops of either -, -, or -ENaC (623). CAP1 largely augments the activity of amiloride-sensitive Na⁺ channels, without altering the number of channels in the plasma membrane (623). CAP1 does regulate ENaC independent of CFTR as demonstrated recently in Xenopus oocytes (272). So far, this autocrine regulatory mechanism has been examined in detail only in the kidney. It will be interesting to learn in future studies about the role of CAP1 in regulating Na⁺ absorption in the colonic epithelium.

In many tissues, expression of ENaC and further proteins participating in epithelial ion transport such as the Na⁺-K⁺-ATPase are upregulated by glucocorticoid and mineralocorticoid hormones. This leads to enhanced Na⁺ absorption and K⁺ secretion (40, 43, 505, 613). Glucocorticoid and mineralocorticoid receptors are present in proximal and distal colon in both surface epithelium and crypts. These steroids exert differential effects on Na⁺ absorption in the colon (25, 535). Low-dose glucocorticoids induce electroneutral Na⁺ absorption in both proximal and distal colon, whereas high concentrations activate both electroneutral and electrogenic absorption, particularly in the distal colon of human and rat (25-27, 91, 158, 512, 613). In contrast, aldosterone induces only electrogenic absorption and inhibits basal electroneutral absorption in the distal colon of rats (27). According to two reports, high concentrations of glucocorticoids antagonize inhibitory effects of aldosterone on electroneutral Na⁺ absorption by binding to aldosterone receptors, thereby further augmenting Na⁺ absorption in rat proximal and distal colon (25, 613). It has been shown that upregulation of electroneutral Na⁺ absorption by glucocorticoids is paralleled by inhibition electrogenic absorption. This may explain why no significant electrogenic Na⁺ absorption is found in the proximal colon, despite the presence of receptors for aldosterone (27). A similar inhibitory effect on mineralocorticoid receptors has been found for the atrial natriuretic peptide (ANP) (534).

Glucocorticoids control epithelial Na⁺ conductance by activation of transcription of ENaC - and -subunits, whereas -ENaC appears to be expressed constitutively (497, 582). Similarly, expression ENaC - and -subunits is also controlled by mineralocorticoids in the adult rat colon. ENaC -subunits are expressed independently of circulating aldosterone (42, 373). Although aldosterone is regulating the expression of ENaC, mRNA levels of ,,-ENaC were surprisingly similar in mineralocorticoid knockout mice, compared with wild-type animals (33, 163). Moreover, although mRNA expression for the three ENaC subunits remained unchanged in these knockout animals, amiloride-sensitive Na⁺ currents were largely reduced in the colonic epithelium (33). These results point to the role of additional proteins regulating Na⁺ absorption, whose expression is also controlled by aldosterone.

The recently discovered serum and glucocorticoid regulated kinase SGK, a member of the serine-threonine kinase family, along with several other mineralocorticoid-regulated genes, is an important regulatory protein in the colon (60, 61, 89, 194, 579, 631). SGK is one of the proteins mediating the early aldosterone action. It is upregulated in kidney collecting ducts and in the colon (638). Principally, aldosterone action can be subdivided into early and late responses. Early aldosterone-regulated gene products occur within 1-3 h after exposure to the steroid hormone, while transcription of ENaC and the Na⁺-K⁺-ATPase is thought to mediate the late response. Other steroids such as estradiol have been demonstrated to affect colonic transport in a nongenomic action (152, 206, 207). This suggests a similar nongenomic action of aldosterone on ENaC (165). However, a rapid induction of the transcription of ENaC - and -subunits within 1 h has been shown to take place in the late distal colon of rat (161). According to these results, increased transcription of - and -ENaC may form part of the early response. Early aldosterone action has been shown to enhance both number and activity of ENaC channels in the cell membrane (202, 411, 579, 631). In renal cells, aldosterone increases the open probability of Na⁺ channels (319).

Other proteins, such as the channel inducing factor (CHIF), have been demonstrated to be transcriptionally controlled by Na⁺ depletion and steroids. CHIF might participate in the regulation of luminal K⁺ channels in the colonic epithelium and the control of K⁺ homeostasis (12, 78, 637). It should be mentioned that upregulation of the activity of other transport proteins like the basolateral Na⁺-K⁺-ATPase by aldosterone is another mechanism by which aldosterone regulates Na⁺ absorption. Moreover, a fast and nongenomic action of aldosterone on Na⁺/H⁺ exchange was detected in the rat distal colon. Nongenomic activation of the Na⁺/H⁺ exchange may occur via an increase in intracellular Ca²⁺ and stimulation of PKC (671). This and other regulatory properties of aldosterone are the subject of a recent review (631).

Apart from the kidneys, the mammalian colon also contributes to the regulation of K⁺ homeostasis by secreting and absorbing K⁺ (515) (Fig. 5). Active K⁺ absorption is restricted to the mammalian distal colon of rat, rabbit, and guinea pig (153, 314, 460). It is mediated by at least two different types of H⁺-K⁺-ATPases, expressed in columnar surface epithelial cells and in the crypts (153). These ATPases are distinguished on the basis of their sensitivity toward ouabain and omeprazole. Although the ouabain-insensitive H⁺-K⁺-ATPase has been cloned, the ouabain-sensitive H⁺-K⁺-ATPase has not yet been identified at the molecular level (1, 128). Both types of H⁺-K⁺-ATPases have been detected in the surface epithelium, whereas the ouabain-sensitive isoform has been identified only in apical membranes of colonic crypt cells. Colonic H⁺-K⁺-ATPases are members of the family of P-type ATPases, similar to the Na⁺-K⁺-ATPase and the gastric H⁺-K⁺-ATPase, consisting of - (HKc) and -subunits (HKc, NaK3). Expression of HKc and NaK is upregulated by dietary Na⁺ and K⁺ depletion, respectively, and by aldosterone (1, 103, 295, 521-523). Interestingly, the luminal ouabain-sensitive H⁺-K⁺-ATPase can function alternatively as a Na⁺-K⁺-ATPase. Therefore, expression of HKc in Xenopus oocytes induces both H⁺-K⁺- as well as Na⁺-K⁺-ATPase activity (103, 108, 488). These results may explain why Na⁺-K⁺-ATPase activity has been detected in previous reports in apical membrane vesicles of colonic epithelial cells (488). Potassium that has been taken up from the luminal side is released to the blood side by basolateral K⁺ channels and probably by the electroneutral KCl cotransporter KCC1 (520).

View larger version (25K): [in this window] [in a new window]   Fig. 5. Cellular model for active K+ absorption in the distal colonic epithelium. K+ is taken up from the luminal side of the epithelium by two different H+-K+-ATPases, one of which is inhibited by ouabain. K+ transport to the blood side of the epithelium may occur by several different mechanisms, including basolateral cAMP- and Ca2+-activated K+ channels and the KCl cotransporter KCC1. Expression of the ouabain-sensitive H+-K+-ATPase is probably limited to the crypt cells.

Transport of SCFA is discussed in this review in regard to its effect on NaCl absorption in the colon. For more detailed information and further aspects on SCFA transport, such as its putative role in colonic carcinoma and ulcerative colitis, we recommend previous reviews (483, 632). In parallel with the absorption of NaCl, SCFA are absorbed by the colonic epithelium (Fig. 6). SCFA are produced during fermentation of dietary fibers by colonic bacteria. Absorbed fatty acids are preferentially metabolized by colonic epithelial cells and exert trophic effects on the epithelium. Absorption of the SCFAs propionate, butyrate, and acetate occurs primarily by nonionic diffusion and paracellular absorption in the proximal colon (387). However, an additional SCFA/HCO exchange mechanism seems to be present in the luminal membrane of rat colonic epithelial cells. This exchanger is distinct from the DIDS-sensitive Cl/HCO antiporter and has not yet been identified at the molecular level (87, 407, 551, 618, 633). Absorption of SCFA not only serves as an additional energy supply for colonic epithelial cells, but has also a significant impact on NaCl absorption (9, 10, 41, 119). SCFA stimulate electroneutral uptake of Na⁺, presumably by acidification of colonocytes and activation of apical Na⁺/H⁺ exchangers (550). Cl absorption is stimulated by increased HCO production during SCFA metabolism and stimulation of the apical Cl/HCO exchanger. Another model has been proposed, in which butyrate is taken up via nonionic diffusion or SCFA/HCO exchange. Subsequently electroneutral NaCl absorption is activated by parallel Cl/butyrate and Na⁺/H⁺ exchange (481). During absorption of SCFA, basolateral volume-sensitive Cl channels are activated, whereas basal and cAMP-activated Cl secretion by CFTR is inhibited (41, 119, 146, 628). It was found that absorption of SCFA depolarizes colonic crypt cells due to cellular acidification and inhibition of K⁺ channels (145). Because HCO secretion to the luminal side of the epithelium is activated, absorption of SCFA has a large impact on the regulation of luminal intestinal pH (95, 618). Thus SCFA transport is an important factor that regulates colonic fluid balance, absorption of NaCl, and luminal as well as cytosolic pH.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Cellular model for absorption of short-chain fatty acids (SCFA), which are metabolized preferentially by colonic epithelial cells. Absorption occurs primarily by nonionic diffusion and paracellular absorption in the proximal colon. Additional SCFA/HCO exchangers are present in the luminal membrane. Butyrate may recycle over the luminal membrane via a Cl/butyrate exchanger. In parallel with a luminal Na+/H+ exchange, this would drive absorption of NaCl. Basolateral K+ channels are inhibited during absorption of SCFA and increase of the intracellular proton concentration, whereas basolateral swelling-induced Cl channels are activated.

	IV. SECRETORY FUNCTION OF THE COLONIC EPITHELIUM

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Another major function of the colon is secretion of electrolytes, which is balanced by absorption. It may facilitate the transport of mucus out of the crypts and maintain hydration of mucus, which is secreted by goblet and columnar epithelial cells in crypts and surface epithelium. Accordingly, mucus secretion is activated by an increase in intracellular cAMP in parallel with electrolyte secretion (248). A limited KCl secretion under resting conditions does become a pronounced KCl/NaCl secretion upon stimulation by secretagogues or when exposed to bacterial toxins. In the absorbing colon and in the absence of secretagogues, release of K⁺ to the luminal side is potential driven and largely maintained by the ENaC. This leads to a luminal K⁺ concentration which is above that of serum. As for the absorption of NaCl, polarized distribution of transport proteins is required for secretory salt transport. Thus secretory epithelial cells contain Cl and K⁺ channels in their luminal membranes, allowing for secretion of KCl. In addition, after secretory stimulation and upon inhibition of absorption, paracellular transport of Na⁺ facilitates secretion of NaCl (228, 323, 404, 536). The apical Cl conductance is formed predominantly by CFTR, which has a central role in colonic ion transport (219). On their basolateral membranes, secretory cells contain Na⁺-2Cl-K⁺ cotransporters that take up Cl from the serosal side of the epithelium together with Na⁺ and K⁺. Basolateral K⁺ channels allow for the recycling of K⁺ via the basolateral membrane, thus hyperpolarizing epithelial cells and maintaining the electrical driving force for Cl secretion. This general scheme (Fig. 7) of electrolyte secretion in the colon has been established many years ago (123) and was originally described in rectal glands of Squalus acanthias (225-227). Water is driven osmotically through the paracellular shunt pathway and is transported via specialized aquaporin water channels (324, 663).

View larger version (33K): [in this window] [in a new window]   Fig. 7. Cellular model for Cl secretion, located predominantly in crypt cells but also in the surface epithelium of proximal and distal colon. Cl secretion is activated by cAMP-dependent stimulation of luminal CFTR Cl channels, which are the predominant if not only luminal Cl channels in the native colon. Cl secretion is paralleled by secretion of K+ via luminal K+ channels and Na+ transport through the paracellular shunt. Cl is taken up into the cells by the basolateral Na+-K+-2Cl cotransporter NKCC1. The rate of secretion is probably controlled by the activity of NKCC1, which is regulated through the intracellular Cl concentration, cell swelling, and probably phosphorylation. Cl transport is maintained by basolateral cAMP-activated KVLQT1/KCNE3 and Ca2+-activated SK4 K+ channels.

Secretion of KCl and NaCl is activated by a whole list of different secretagogues, which have been summarized elsewhere (22, 645). These secretagogues act via different intracellular messengers that are outlined below (141, 221, 228, 323, 404, 536) (Fig. 8). Coordinated action of apical and basolateral ion channels, together with basolateral cotransporters and the Na⁺-K⁺-ATPase, is essential. Thus, during secretion, Cl uptake from the basolateral side has to keep up with luminal Cl exit. Also, depolarization of cells by opening of luminal Cl channels has to be compensated by activation of basolateral K⁺ channels to maintain the electrical driving force for luminal Cl exit (136, 219, 221).

View larger version (36K): [in this window] [in a new window]   Fig. 8. Control of electrolyte secretion in the colon. Increases in the intracellular second messengers cAMP, Ca2+, cGMP, protein kinase C (PKC), and calmodulin-dependent kinase (CAMK) stimulate secretion of KCl and NaCl by activation of transport proteins in luminal (CFTR Cl channels, K+ channels) and basolateral (Na+-2Cl-K+ cotransporter, K+ channels, Na+-K+-ATPase) membranes. Second messenger pathways are activated and inhibited, respectively, by a large number of hormones and neurotransmitters. Only a few important stimulatory hormones and neurotransmitters are shown, such as vasoactive intestinal polypeptide (VIP), acetylcholine (ACh), histamine, secretin, leukotrienes, serotonin, adenosine, and nitric oxide (NO). Some of these hormones, for example, VIP and NO, also control blood supply of the intestinal epithelium. An incomplete list of hormones that antagonize secretion is shown, including neuropeptide Y (NPY), somatostatin, opiates, norepinephrine (NOR), and autocrine survival factor (ASF). Hormones and neurotransmitters are released from capillaries, by enteric nerves, or immune cells such as mast cells and lymphocytes. In addition, other factors are secreted in paracrine or autocrine fashion. Food components, bile acids, and bacterial or viral toxins may also exert secretory effects on the colonic mucosa.