Epithelial Cl-channels

This area of research has been reviewed by Gogelein [9] and myself [10]. Three general classes of CP-channels have been found. 

Large conductance CP-channels were described for renal epithelial cells such as MDCK-cells, urinary bladder, collecting duct and A6-cells [51-54] and in pulmonary alveolar cells [55]. 

The physiological role of the ICOR is not clear and may be heterogeneous in the various tissues. In the thick ascending limb of the loop of Henle this channel appears to serve as the exit for CP at the basal cell pole [16,65,66], This conductive mechanism, therefore, is required for the reabsorption of Na and CP by this segment of the nephron [16]. In the rectal gland of Squalus acanthias a very similar channel is utilized for Na and CP secretion. In these latter cells the CP-channel is present in the luminal membrane and is controlled by cytosolic cAMP [15,56,71]. It has been claimed that this kind of channel is also responsible for the secretion of CP in the colonic crypt cell, in colonic carcinoma cells and in respiratory epithelial cells [17,19,20,22]. Recent data have cast some doubt on this concept  

CP-channels with smaller conductance have first been noted in the rectal gland of Squalus acanthias by ourselves and in the colonic carcinoma cell line HT29 [61,73]. Later these types of 5-15 pS CP-channels were also found in pancreatic ducts, A6-cells and many other cells [74,75]. It is now claimed that this kind of channel is much more relevant than the ICOR for the pathophysiology of cystic fibrosis [12]. 

CP-channels with even smaller conductance have been described for the lacrimal and other exocrine glands [76,77]. These channels have a conductance of 1-2 pS. Unlike the ICOR-channel they appear to be blocked by millimolar concentrations of furosemide [77]. Most recent and only partially published data from my own laboratory obtained with the above modified nystatin technique [50,133,134] indicate that the respiratory epithelial cells and colonic carcinoma cells possess these types of small CP channels, and that these channels are involved in hormonal regulation of CP-conductance (cf. section 5). These CP-channels are regulated by cytosolic Ca. Hormonally induced increases in cytosolic Ca lead to an abrupt increase in the probability of these small CP-channels being open, yet they have no effect on the ICOR-channel. Data of this kind reinforce that the physiological importance of these small CP-channels may have been grossly underestimated. 

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The regulation of epithelial Cl -channels has been examined in many laboratories, and these studies have been intensified with the recognition that the Cl -channel regulation in epithelia is defective in a very common and clinically serious inherited disease, namely cystic fibrosis [17,18]. Efforts in this direction have not yet arrived at coherent concepts, and many published models [19-22] may have to be modified or revised. 

The receptor-operated Cl -channels of the central nervous system (CNS) are gated by the respective agonists GABA and glycine. Most Cl -channels can be inhibited by disulphonate stilbenes. Muscle Cl -channels can be inhibited by anthracene-9-carboxylate (A9C) and probably by IAA-94. The ICOR Cl -channel is fairly sensitive to NPPB. It should be noted, however, that none of these probes, except for the GABA- and glycine-receptor Cl -channels, is of sufficient affinity and selectivity to permit the channel identification by its use. This dilemma is one of the reasons why the purification of epithelial Cl -channels lags behind that of the CNS Cl -channels. 

Other blockers of epithelial Cl -channels are of the aryl-amino-benzoate type or phenoxy-acetic-acid type [70]. Very few systematic surveys comparing different classes of blockers in one type of Cl -channel are available at this stage. One such study has been performed in membrane vesicles from kidney cortex [80]. In this study IAA-94 and NPPB (cf. Fig. 2) turned out to be the most potent blocker of conductive Cl -flux. In another systematic survey the Cl -conductance of the sweat duct was examined, and it was found that dichloro-DPC (Fig. 2) was the most potent inhibitor of the transepithelial Cl -conductance [90]. 

Fourteen-membered ring macrolides can inhibit Cl secretion across the airway epithelial Cl channel. Although the subcellular mechanism of this action warrants further studies, the inhibition of Cl secretion may lead to the reduction of liquid secretion across the airway mucosa toward the lumen. It is thus likely that the favorable effects of 14-membered macrolides on chronic airway hypersecretion might be related, at least in part, to the action on airway epithelial Cl channel. 

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), a chloride (CF) channel characterised by chloride permeability and secretion, and also by the regulation of other epithelial ion channels (Eidelman et al, 2001). Mutations in the CFTR gene lead to an impaired or absent Cl conductance in the epithelial apical membrane, which leads to defective Cl secretion and absorption across the epithelium. Genistein (Illek et al, 1995 Weinreich et al, 1997) and other flavonoids (Illek and Fisher, 1998) have been shown, in different animal and tissue models, to activate wild-type CFTR and CFTR mutants by (Eidelman et al, 2001 Roomans, 2001 Suaud et al, 2002) ... 

In many epithelia Cl is transported transcellularly. Cl is taken up by secondary or tertiary active processes such as Na 2Cl K -cotransport, Na Cl -cotransport, HCOJ-Cl -exchange and other systems across one cell membrane and leaves the epithelial cell across the other membrane via Cl -channels. The driving force for Cl -exit is provided by the Cl -uptake mechanism. The Cl -activity, unlike that in excitable cells, is clearly above the Nernst potential [15,16], and the driving force for Cl -exit amounts to some 2(f-40mV. 

The present chapter will address the following issues (1) a very brief overview on the properties of the different types of Cl -channels in the various mammalian cells (2) a short summary on what is known of Cl channels on a molecular basis (3) a discussion of pharmacological agents blocking the various Cl -channels and (4) a specific section dealing with the regulation of epithelial and maybe other Cl -channels. This entire area has been reviewed rather extensively in the recent past. A large number of references will be provided in order to keep this text concise. The entire field of Cl -channels in the central nervous system will only be touched upon to compare these channels to the Cl -channels in apolar cells and epithelia. 

It has been known for some time that the Cl -conductance of epithelial cells can, in addition to its regulation via cAMP, be enhanced by increases in cytosolic Ca " (cf. Fig. 3). This has been shown with Ca -ionophores [120,121] or with hormones increasing cytosolic Ca such as carbachol, neurotensin, ATP, etc. [50,103,104]. Usually these agonists have dual effects. They increase the Cl - as well as the K" -conductance [104]. Stubs et al. [122] have shown that CF cells still increase their Cl -conductance in response to ATP. Another mechanism of Cl -channel activation has been described in whole-cell patches of colonic carcinoma and RE cells [123,124] when the cells are exposed to hypotonic media they swell and increase their Cl -conductance. This is a rather general phenomenon which is present in a lot of cells [11]. In their effort to reduce cell volume in hypotonic media (regulatory... 

Recent work in our laboratory (Kompella, Mathias, and Lee, unpublished observation) has revealed that activation of the cAMP-regulated Cl channels in the conjunctiva also enhances the transcytosis of horseradish peroxidase. 8-Bromo-cAMP (a membrane-permeable analog of cAMP) and terbutaline (a p2-adrenergic agonist known to increase intracellular levels of cAMP in other epithelial tissues [238]), at 0.5 mM, were found to enhance the transport of 100 pg/ mL HRP from the mucosal side to the serosal side of the pigmented rabbit conjunctiva by a factor of 4 (Fig. 11). 

Shimizu, T., Morishima, S. and Okada, Y., 2000, Ca2+-sensing receptor-mediated regulation of volume-sensitive Cl- channels in human epithelial cells. J Physiol 528, 457-72. 

The chloride channel is vital for proper absorption of salt (NaCl) and water across the plasma membranes of the epithelial cells that line ducts and tubes in tissues such as lungs, liver, small intestine, and sweat glands. Chloride transport occurs when signal molecules open CFTR Cl channels in the apical (top) membrane surface of epithelial cells. In CF the failure of CFTR channels results in the retention of Cl within the cells. A thick mucus or other secretion forms because osmotic pressure causes the excessive uptake of water. The most obvious features of CF are lung disease (obstructed air flow and chronic bacterial infections), and pancreatic insufficiency (impaired production of digestive enzymes that can result in severe nutritional deficits). In the majority of CF patients, CFTR is defective because of a deletion mutation at Phe508, which causes... 

The flux of Na+, K+, and CF from the lumen into the epithelial cells in the TAL is mediated by the Na+-K -2C1" symporter (Figure 28-1). This symporter captures the free energy in the Na" electrochemical gradient established by the basolateral Na+ pump and provides for uphiU transport of K+ and Cl" into the cell. Hyperpolaiization of the luminal membrane due to conductance through apical K+ channels (called ROMK) and depolarization of the basolateral membrane due to Cl" conductance through basolateral Cl" channels (called CLC-Kb) result in a transepitheUal potential difference, with the lumen positive with respect to the interstitial space. This lumen-positive potential difference repels cations (Na+, Ca +, and Mg +) and thereby provides an important driving force for the paracellular flux of these cations into the interstitial space. 

Weber AJ, Soong G, Bryan R, Saba S, Prince A. Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl-channel function. Am J Physiol Lung Cell Mol Physiol 2001 281 L71. 

In respiratory epithelial (RE) cells the Cl -conductance was attributed to the ICOR channel. In fact, it was reported by Frizzell et al. and Welsh s laboratories that catecholamines increased the incidence of ICOR channels in cell attached patches of normal RE cells but failed to do so in CF cells [110,111], Later both laboratories presented data on excised membrane patches of RE cells in which the protein kinase A which was added to the cytosolic side produced ICOR channel activity in the normal cells but not in the CF tissues [19,20]. This finding was reproduced by Guggino and coworkers [22] for RE cells and by others for lymphocytes [46]. Protein kinase C at physiological Ca -activities had a comparable effect in normal cells but also failed to function in CF cells [22,112]. 

There is growing evidence implicating Na+-dependent solute transporters and intracellular as well as extracellular Ca2+ in the physiological regulation of the paracellular pathway [81,203,204], Such modulation of paracellular permeability is especially important for drugs such as peptides and oligonucleotides that exhibit poor permeability characteristics across both the cornea and the conjunctiva [150,152,154,155], In addition, ion transporters such as Cl and Ca2+ channels have been implicated in macromolecular transport (see Sections IV.B.2 and IV.B.4). In the following discussion, some key ion transport processes and their possible roles in solute transport across epithelial tissues are summarized. 

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