[45]
CHLORIDE CHANNEL BLOCKERS
[45] C h l o r i d e
Channel
793
Blockers
By RAINER GREGER
After a brief introduction into the chloride-transporting membrane proteins, this chapter will focus on a new class of chloride channel blockers. It will be shown that these substances belong to a larger family of agents, of which so-caUed loop diuretics are just one member. Introduction This contribution restricts itself to the blocking of chloride channels and not to chloride permeation in general. The ability to distinguish between permeability in general and permeation through a channel depends on the methods used. If radiolabeled chloride is the measured signal, all permeation pathways for chloride existing in the membrane under study will contribute to the signal. If the chloride flux is measured electrically, only systems transporting chloride with its negative charge will be monitored. It should be clear that this latter chloride flux should only be called "electrogenic," and it cannot, a priori, be equated with the flux of chloride through ionic channels. Fortunately, this distinction appears puristic from a practical point of view since only very few rheogenic chloridetransporting carrier systems have been described thus far, whereas chloride channels are present in many different cell types. In the last 10 years or so an identification of chloride channels has become possible by the patchclamp technique.t Still, however, most of our knowledge about blockers of chloride current comes from electrophysiological studies where macroscopic currents or voltages have been measured. In this chapter only chloride transport systems and, more specifically, chloride channels in a few well-defined preparations, mostly of vertebrate offgin, are considered. Chloride channel blockers with very little molecular modification, or generally at higher concentrations, also inhibit other chloride-transporting proteins. This finding has two ramifications on which I will expand. (1) Blockers are usually far less specific than one wishes them to be, and much caution is needed to deduce the existence of a transport system solely on the basis of inhibitor interaction. This consideration, as trivial as it is, is still ignored frequently, and the simple rule that a pharmacological effect must always be looked at on the basis of dose-response curves is neglected in many reports. (2) The fact that a blocker interferes with different affinit B. S a k m a n n a n d E. Neher, "Single-Channel Recording," P l e n u m , N e w York, 1983.
METHODS IN ENZYMOLOGY, VOL. 191
Copyright© 1990by AcademicPress,Inc. All rightsof reproduction in any form reserved.
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PHARMACOLOGICALAGENTS IN EPITHELIAL TRANSPORT
[45]
ties with several chloride-transporting epithelia may also indicate that the transport proteins, even though they may appear functionally different, may share in common some part(s) of the molecule. Therefore, I will briefly describe various chloride transport systems. The bicarbonate/chloride exchange system is present in the red cell membrane in the form of the band 3 protein (for a review, see Passow2). This system or modifications of this system are found in many cells, and may serve pH regulation, 3 bicarbonate transport (for a review, see Novak and Greger4), or chloride transport. 5 Another, recently detected6 chloride transport system is the Na+/2C1-/K + carrier. This system is present in many polar and apolar cells (for a review see Ellory et aL7). It is responsible for the cellular uptake of chloride. If present in epithelia, s it serves the transepithelial transport of NaCI, and is usually found together with chloride channels on the opposite cell membranes. 9 Historically, this Na+/2CI-/K ÷ carrier system is remarkable since blockers for this system, the loop diuretics of furosemide type, had been known for more than 20 years before the system itself was recognized. In fact, the loop diuretic-sensitive transport system was long believed to be an energy-dependent chloride pump. Whereas it is clear now that what had been believed to be a primary pump sensitive to furosemide-like substances turned out to be a secondarily active chloride carrier, chloride pumps are still postulated for insect epithelia) ° Another chloride-dependent carrier system has been recently detected, namely a chloride/formate exchange.11
G e n e r a l P r o p e r t i e s of Chloride C h a n n e l s The introduction of the patch-clamp technique has now made it possible to study individual chloride channels, to measure their conductances, permselectivities, opening kinetics, activation mechanisms, and the mechanism of action of inhibitors (for a review, see GOgelein12). Models for the 2 H. Passow,Rev. Physiol. Biochem. Pharmacol. 103, 61 (1986). a R. C. Thomas, Curt. Top. Membr. Transp. 13, (1980). 4 I. Novak and R. Greger, Pfluegers Arch. 411, 546 (1988). 5C. M. Liedtkeand U. Hopfer,Am. £ Physiol. 242, G272 (1982). P. Geck, C. Pietrzyk, B.-C. Burekhardt, B. Pfeiffer,and E. Heinz, Biochim. Biophys. Acta 600, 432 (1980). 7j. C. Ellory, P. B. Dunham, P. J. Logue, and G. W. Stewart,Philos. Trans. R. Soc. London, B 299, 483 (1982). s R. Greger, PhysioL Rev. 65, 760 (1985). 9 R. Greger, E. Sehlatter, and H. G0gelein, News in Physiol. Sci. 1, 134 (1986). ~oG. A. Gereneser, in "Chloride Transport Couplingin BiologicalMembranesand Epithelia" (G. A. Gereneser, ed.), p. 183.Elsevier,Amsterdam, 1984. H L. Schild, G. Giebiseh, L. Karniski, and P. S. Aronson, PfuegersArch. 4117,156 (1986). ~2H. Grgelein, Biochim. Biophys. Acta 947, 521 (1988).
[45]
CHLORIDE CHANNEL BLOCKERS
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channels have been derived from such data. At the same time, ligands are used to purify certain chloride channel proteins and to sequence their amino acids. This area of research has exploded during the last few years. I will restrict myself to summarizing the published work on single channels merely in table form. Arbitrarily, at least at this point, the chloride channels may be divided into those of neuronal origin, those present in smooth and striated muscle, and those found in the various epithelia. As is apparent from Table I, 13-42 it is difficult to find common denominators for the various channels. For example, the single-channel slope conductance varies between less than 1 and 500 pS. Even if one considers that the composition of the bathing solutions may have been different, this range appears surprising. One possible clue to the origin of such a wide range may be the fact that chloride channels appear in clusters within one patch. Such clusters can be taken as subunits of one single channel, which then would have a rather large unitary conductance. Alternatively, clusters t3 D. G. Owen, M. Segal, and J. L. Barker, Nature (London) 311, 567 (1984). t40. P. Hamill, J. Bormann, and B. Sakmann, Nature (London) 305, 805 (1983). t~ R. N. McBarney, S. M. Smith, and R. Zorec, J. Physiol. (London) 367, 68P (1985). ~6A. J. Martin and M. R. Gold, Biophys. J. 41, 62 (1983). t7 V. I. Geletyuk and V. N. Kazachenko, J. Membr. Biol. 86, 9 (1985). t8 D. Chesnoy-Marchais and M. G. Evans, J. Physiol. (London) 357, 64P (1984). ~9M. M. White and C. Miller, J. Biol. Chem. 254, 10161 (1979). 2op. T. A. Gray, S. Bevan, and J. M. Ritchie, Proc. R. Soc. London, B 221, 395 (1984). 2~p. T. A. Gray and J. M. Ritchie, Proc. R. Soc. London, B 228, 267 (1986). 22S. S. Kolesnikov, A. L. Lyubatsky, and E. E. Fesenko, Vision Res. 24, 1295 (1984). 23A. L. Blatz and K. L. Magleby, Biophys. J. 43, 237 (1983). 24A. L. Blatz and K. L. Magleby, Biophys. J. 47, 119 (1985). 25W. Schwarze and H.-A. Kolb, PfluegersArch. 402, 281 (1984). 26A. Coulombe and H. Duclohier, J. Physiol. (London) 350, 52P (1984). 27 R. Shoemaker, J. Naftel, and J. Farley, Biophys. J. 47, 465a (1985). 2s G. P. H. Young, J. D.-E. Young, A. K. Deshpande, M. Goldstein, S. S. Koide, and Z. A. Cohn, Proc. NatL Acad. Sci. U.S.A. 81, 5155 (1984). 29D. J. Nelson, J. M. Tang, and L. G. Palmer, J. Membr. Biol. 80, 81 (1984). 3oH. A. Kolb, C. D. A. Brown, and H. Murer, PfluegersArch. 403, 262 (1985). 31 R. Greger, Physiol. Aktuel. 2, 47 (1986). 32R. Greger, M. Bleich, and E. Schlatter, Renal Physiol. Biochem. 13, 37-50 (1990). 33H. GOgeleinand R. Greger, PfluegersArch. 407 (Suppl. 2), S142 (1986). W. Hanrahan, W. P. Alles, and S. A. Lewis, £ Gen. Physiol. 84, 30a (1984). 3s A. Marty, Y. P. Tan, and A. Trautmann, J. Physiol. (London) 357, 293 (1984). 36I. Findlay and O. H. Petersen, Pfluegers Arch. 403, 328 (1985). 37R. Grager, E. Schlatter, and H. GOgelein,Pfluegers Arch. 409, 114 (1987). 3s H. G6gelein, E. Schlatter, and R. Greger, Pfluegers Arch. 409, 122 (1987). 39M. J. Welsh, PfluegersArch. 407 (Suppl. 2), S116 (1986). 4oj. p. Hayslett, H. G6gelein, K. Kunz¢lmann, and R. Greger, Pfluegers Arch. 410, 487 (1987). 4t R. Greger and K. Kunzelmann, in "Epithelial Secretion of Water and Electrolytes" (J. A. Young and P. D. Wong, eds.). Springer-Verlag, New York, 1989. 42M. E. Krouse, G. T. Schneider, and P. W. Gage, Nature (London) 319, 58 (1986).
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PHARMACOLOGICAL AGENTS IN EPITHELIAL TRANSPORT
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are interpreted as individual channels, which then leads to rather small values for the unitary conductance. According to accepted theory, a distinction between both alternative explanations can be made on the basis of whether the single conductance events (or sublevels) are dependent on or independent of each other as tested by statistical methods. In case of independence of events, the term subunit would be misleading if it was not qualified as independent subunit. If the conductance events are dependent on each other, independent single channels are excluded and the term subunit is appropriate. The situation may be even more complex if one looks closely at original recordings. Frequently, substates of differing current amplitude are observed, and even one individual chloride channel may vary its slope conductance to some extent as the experiment proceeds. To make it even worse, in one patch several different chloride channels can be found. This has been recognized in myotubes,23~ and in epithelia. ~2,aS,a7,3sUsually, large and small channels are distinguished, but if there were no additional criteria for distinction, the slope conductance alone would clearly not be sufficient. Finally, it should not be ignored that the patch-clamp technique for the recording of single ionic channels has its restrictions. It will "see" a channel only if it is open for some limited time and at some limited current level. Our routine conditions (signal-to-noise ratio for a filter frequency of 0 . 5 - l kHz) would set the limit such that a channel will be seen only if it is open at least some 0.2-0.5 msec and if it has a conductance above 5 pS. Thus, in single-channel recordings, we will ignore any very small chloride channel whether it was physiologically relevant or not. It should be clear, however, that even very small channels can be found in whole cell recordings. To summarize this issue, it seems very dangerous to classify chloride channels according to their conductance. Inherent pitfalls include the substate and subunit dilemma, and, probably even more serious, the variability of the conductance for otherwise identical channels. A similar dilemma in classification of chloride channels is apparent if one considers the heterogeneity of the voltage dependence. Three patterns are found with almost equal frequency: (l) increase in open probability by depolarization, (2) increase in open probability by hyperpolarization, and (3) bell-shaped curves with the maximum around 0 mV clamp potential. These different patterns cannot be ascribed to the three subclasses of channels chosen for this table. In fact, for neuronal chloride channels all three patterns have been reported. For the epithelial chloride channels it seems to be the "rule" that depolarization increases the open probability and, in many instances, also the slope conductance. Where determined, the listed channels are clearly anion selective, and large anions are usually excluded. At least in some types of channels I- and
[45]
CHLORIDE CHANNEL BLOCKERS
799
to a smaller extent Br- are excluded. Chloride channels may be regulated by Ca 2+, but the calcium dependence is also heterogeneous and ranges from Ca 2+ dependent to Ca2+ inhibited. However, as with selectivity, too few data have been reported to arrive at any firm conclusion. To summarize Table I, I should like to conclude that chloride channels in the various cells show a large variability. In this respect, other ionic channels, e.g., the acetylcholine receptor, the Na + channels, and the K + channels, seem to be more homogeneous. As devastating as the data in Table I may be, I am optimistic that subclasses of channels are emerging with far less heterogeneity, as, for example, the various chloride channels in secretory epithelia. Most research groups would agree that the cAMP-dependent chloride channels present in these epithelia have a narrow conductance range (around 40-50 pS), a similar selectivity, and a comparable voltage dependence. Chloride C h a n n e l Blockers According to their targets, the various agents may be grouped into substances interfering with the 7-aminobutyfic acid (GABA) and glycine receptor chloride channels, and agents which interfere with chloride channels in muscle membrane, apolar cells such as the red blood cell, and in epithelial cells. This type of classification is not at all satisfactory since, on the one hand, some agents appear to act in several of the above-described channel types. On the other hand, the representatives for one group of blockers act quite differently. Some may interfere with the activation of the channel, e.g., GABA antagonists, others may act at the chloride-binding site, and yet others are not at all defined in their mode of action. Therefore, this chapter will list the different blockers, provide reference to pertinent recent work, but will not go into details of the mechanism of blocking. For one newly detected group of agents, namely the arylaminobenzoates, sufficient data are available to discuss some hypothetical views of the mechanism of interaction.
Agents Acting at the Neuronal Chloride Channels Well-studied examples are the GABA receptor and the glycine receptor chloride channels. GABA binding gates the former channel and that of glycine the latter. 14 The GABA interaction is modified by diazepam and related agonists (central type of receptor) such that the GABA effect is enhanced. Diazepam antagonists have the opposite effect and, therefore, reduce the GABA effect on the chloride current. These substances then act as convulsants. It is interesting to note that shortly after their detection in
800
PHARMACOLOGICAL AGENTS IN EPITHELIAL TRANSPORT
[45]
the brain, peripheral diazepam receptors were also found. The function of these receptors, e.g., in the nephron43 is still obscure. This appears puzzling as the benzodiazepam binding coincides with the site of active NaCl reabsorption in the nephron. This has prompted us to examine several of these agonists and antagonists (kindly provided by Dr. P. Skolnick) in isolated perfused thick ascending limb segments of rabbit and mouse kidney. We are unable to demonstrate an effect on active NaC1 reabsorption (unpublished observations). Other, and chemically not at all related, substances have been reported as inhibitors of the GABA-activated chloride channel: pentylenetetrazole,~ picrotoxin and bicuculline,45 and bicyclic phosphates and several insecticides. ~ The glycine-operated chloride channel is inhibited by strychnine and related compounds. In fact, these antagonists have been used to purify this channel protein. 47
Agents Acting at the Muscular Chloride Channel No experiments are available at the level of single chloride channels (patch-clamp analysis). Therefore, the data on putative blockers of chloride fluxes may reflect the properties of systems other than chloride channels. In the striated barnacle muscle cAMP-stimulated chloride fluxes were inhibited by rather low doses of sulfonic acid stilbene derivatives4S: 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This result documents the similarity of this chloride transport system in muscle cell membrane to that in the red blood cell (vide infra). Also, furosemide in a rather high dose (0.6 mmol/liter) was effective as a reversible inhibitor in this muscle preparation. As will be pointed out later, it is not pertinent, and may even be incorrect, to conclude from this finding that chloride transport occurred via the Na+/2C1-/K+ cotransport system. A recent review49 summarizes the work with chloride channel blockers in striated muscle and subdivides the agents into four groups: (1) polycyclics, with anthracene-9-carboxylate as one example, (2) benzoates, with furosemide as an example, (3) phenoxyacetates, with ethaerynic acid as 43 D. Butlen, FEBSLett. 169, 138 (1984). 44 B. A. Weissmann, J. Cott, D. Hommer, R. Quirion, S. Paul, and P. Skolnick, in "Benzodiazepine Recognition Site Ligands: Biochemistry and Pharmacology" (G. Biggio and E. Costa, eds.), p. 139. Raven, New York, 1983. 45 K. G. Thampy and E. M. Barnes, Jr., J. Biol. Chem. 259, 1753 (1984). 46 j. R. Bloomquist and D. M. Soderlund, Biochem. Biophys. Res. Commun. 133, 37 (1985). 47 D. Graham, F. Pfeiffer, and H. Betz, Eur. J. Biochem. 131, 519 (1983). 48j. M. Russel and M. S. Brodwick, J. Gen. Physiol. 78, 499 (1981). 49A. H. Bretag, Physiol. Rev. 67, 618 (1987).
[45]
CHLORIDE CHANNEL BLOCKERS
80 1
one example, and (4) sulfonates and sulfamides, with disulfonic stilbenes as an example. It is difficult to accept the first three groups as different entities, since it will be shown below that these agents have far more in common than may be apparent at first glance. The methodology, as it has been applied to date, does not permit any conclusion as to how these substances reduce chloride fluxes or chloride conductance. However, recent studies with these substances in epithelial chloride channels5° (cf. next section) may also be used to extrapolate these data to the muscle chloride channel. In the smooth muscle cell most evidence favors the view that cytosolic chloride activity is above Nernstian equilibrium. 51 Many data are available on anion replacements on the extracellular compartment and their effect on membrane voltage, as are many data on disulfonate stilbene as well as on furosemide effects. Little, however, is known on the properties of the respective chloride channels. 27 It has been argued that SITS or DIDS, in analogy to their effects in Torpedo electric organ chloride channel, 52 may also interfere with chloride channels in smooth muscle cells. Also, the effects of organic "nitro" compounds, such as nitroglycerin, have been ascribed to the interference of these compounds with chloride channels. 51
Agents Acting in Apolar Cells One specific, but obviously very complex, chloride transport system is that of the red blood cell. There, the vast majority of the chloride flux occurs via the band 3 protein. 2 However, chloride channels are also present in these cells. A recent survey has compared several classes of agents in their ability to block the anion shift via the band 3 protein and the chloride movement through single chloride channels. 53 It was concluded that both routes of chloride movement take the same pathway, namely, via the band 3 protein. In other words, this protein may perform electroneutral anion exchange (quantitatively most important), and may also permit the channeling of chloride ions. From this point of view, older data using furosemide 2 and flufenamic aci@4 may be looked at from a new perspective. Flufenamic acid and related substances belong to the group of chloride channel blockers, cf below. The band 3 protein may thus bind blockers of three different classes: the sulfonic acid stilbene derivatives, compounds related to furosemide, and compounds related of the arylaminobenzoate so p. Wangemann, M. Wittner, A. Di Stefano, H. C. Englert, H. J. Lang,E. Schlatter, and R. Greger, Pfluegers Arch, 407 (Suppl. 2), S128 (1986). 5~ V. A. W. Kreye and F. W. Ziegler, Adv. Microcirc. 11, 114 (1982). 52C. Miller and M. M. White, Proc. Natl. Acad. Sci. U.S.A. 81, 2772 (1984). 53 W. Schwarz and H. Passow, Proc. Int. Union Physiol. Sci. 30th Congr. 16, 544 (1986). 54J.-L. Cousin and R. Motais, Biochim. Biophys. Acta 687, 156 (1982).
802
PHARMACOLOGICAL AGENTS IN EPITHELIAL TRANSPORT
[45]
class. It is important to realize that the affinities to these three classes of substances are quite different from those needed in other preparations. Micromolar concentrations are needed to block the band 3 protein by sulfonic acid stilbenes, yet much higher concentrations are usually required in other systems. Conversely, the concentrations needed of furosemide and even more so of bumetanide are much higher for the band 3 protein when compared to that needed to interfere with the Na+/2CI-/K + cartier system,s Similarly, rather high concentrations of arylaminobenzoates are necessary to inhibit the band 3 protein. A great deal of effort has been directed toward the pharmacology of glial cell volume regulation, and it was recognized some 4 years ~tgo that several transport systems in which chloride participates are probably responsible for the glial volume increase, which in turn may be the pathophysiological basis of traumatic brain edema. In a recent study, several classes of substances have been tested in brain slices and it was found that the most effective inhibitors were derived from the phenoxyacetic acid group. Specifically (2,3,9,9a-tetrahydro-3-oxo-9ot-substituted-lH-fluoren7-yl)oxyalkanoic acids were effective in doses as low as 10-12 mol/liter. However, the authors 55 concluded only that this class of inhibitors blocks C1- uptake and have no evidence that this uptake is via chloride channels.
Agents Acting in Epithelia Soon after the recognition of the existence of chloride channels in epithelia such as the thick ascending limb, the colon, and the trachea, a search for agents blocking these channels was initiated in our laboratory. The focus was on anthracene-9-carboxylate and diphenylamine-2-carboxylate since these two substances, previously used in the striated muscle chloride channel 5n and in the red blood cell,54 showed some inhibitory activity on the chloride conductive pathways in the thick ascending limb 57 and in the amphibian diluting segment.5s Our survey covered a broad spectrum of substances and used a more systematic approach for the substances showing strong activity. The bioassay used in this survey, namely, the in vitro perfused thick ascending limb of the loop of Henle, has the great advantage that chloride conductance is present only at the blood side of the cell, whereas the Na+/2CI-/K + carrier is present only in the 55E. J. Cragoe, Jr., O. W. Woltersdoff, Jr., N. P. Gould, A. M. Pietruszkiewie-z, C. Ziegler, Y. Sakurai, G. E. Stokker, P. S. Anderson, R. S. Bourke, H. K. Kimelberg, L. R. Nelson, K. D. Barron, J. R. Rose, D. Szarowski, A. J. Popp, and J. B. Waldman, J. Med. Chem. 29, 825 (1986). 56 p. T. Palade and R. L. Barchi, J. Gen. Physiol. 69, 879 (1977). 57A. Di Stefano, M. Wittner, E. Schlatter, H. J. l_ang, H. Englert, and R. Greger, Pfluegers Arch. 405 (Suppl. 1), $95 (1985). 5s H. Oberleithner, M. Ritter, F. Lang, and W. Guggino, Pfluegers Arch. 398, 172 (1983).
[45]
CHLORIDE CHANNEL BLOCKERS
803
lumenal membrane) Loop diuretics of the furosemide type interfere with this carder system but have little effect on chloride channels. A simple experiment leads to this distinction: whereas loop diuretics inhibit the active transport of chloride and sodium instantaneously when added to the lumenal perfusate, they are devoid of effect when added to the basolateral perfusate. Conversely, blockers of the arylaminobenzoate type, as they are listed in Table II, block the active transport of chloride at low concentrations when added to the basolateral perfusate but not when added at the same concentration to the lumenal perfusate. Nevertheless, if the furosemide concentration on the basolateral cell pole is increased to very high concentrations of 10-a tool/liter or more, some inhibition will also be observed. Similarly, if the chloride channel blocker is added to the lumenal perfusate at very high concentration, it may also exert an effect from this side. These effects may be due to a diffusion of furosemide or the chloride channel blocker to the other cell membrane or to effects other than that on the Na+/2C1-/K + carder or on the chloride channel. The first explanation becomes more likely the higher the lipid solubility of the tested compound is. In case of furosemide and related compounds, the lipid solubility at physiologic pH is rather low, but for the chloride channel blockers the opposite is true. Some 30-80% distribute into the lipid phase (e.g., CH2CI2) at pH 7.4. It is not surprising then that these substances also act usually at 10-100 times higher concentrations from the lumenal cell side. 5° The second explanation given above comprises a heterogeneous group of possible effects. Of course, it must be considered that one individual substance may not only inhibit chloride channels, but may also interact with the Na+/2C1-/K + carrier. We have several examples available. 59,6° It should be clear that preparations other than the intact epithelium, e.g., the isolated membrane patch, must be used to prove this potential effect of an Na+/2C1-/K + carrier blocker on the chloride channel. Another potential complication comes from the fact that the tested compound may interfere with other systems. For high concentrations of furosemide and related compounds, an interference with carbonic anhydrase has been shown.61 It should also be noted that the loop diuretics of furosemide type have originated from studies with carbonic anhydrase inhibitors. Furthermore, lipophilic anions will also find access to the mitochondria and will short circuit pH gradients across thinner mitochondrial membrane, thus inter59 R. Greger, P. Wangemann, M. Wittner, A. Di Stefano, H. J. Lang, and H. C. Englert, in "International Conference on Diuretics, Sorento, 1986" (T. Andreucci, ed.), p. 33. Nijhoff, Boston, Massachusetts, 1987. 6o R. Greger, H. J. Lang, H. C. Englert, and P. Wangemann, in "Diuretics" (J. Puschett, ed.), p. 131. Elsevier, New York, 1987. 6~ T. H. Maren, "Renal Physiology: Men and Ideas." Am. Physiol. Sot., Bethesda, Maryland, 1987.
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feting with mitochondrial function. This putative mechanism may be responsible for the poorly reversible inhibitory effect seen with arylaminobenzoates at high concentrations. This effect is usually followed by a cell depolarization. 5° The chloride channel blockers of the arylaminobenzoate type are also known to block cyclooxygenase,~2 and this may generate a dilemma in tissues in which the chloride conductance is increased by prostaglandins. Hence, the reduction in chloride conductance observed in airway epithelial cells after the addition of very high concentrations of diphenylamine-2-carboxylate39 may, in part, be caused by cyclooxygenase inhibition. 63 From our studies we have determined that furosemide is not a chloride channel blocker, u and this contrasts to other studies on the cornea65 and on the tear gland, u It should be noted, however, that in the intact preparation, furosemide will lead to a reduction of the chloride conductance simply because it reduces cytosolic chloride activity,s In isolated chloride channels, we have never observed any effect of furosemide.37,u Table III summarizes several potent chloride channel blockers as documented by their inhibitory effect on the chloride conductance in isolated perfused thick ascending limbs. Some of these compounds have also been tested in isolated chloride channels of the rectal gland, a7 in airway epithelial cells, u in the thick ascending limb of the loop of Henle, 32 and in colonic carcinoma cells?° It is apparent that all listed compounds possess several common features. All are anionic at physiological pH. The anionic group is a carboxylate. All compounds possess an amino group, and all compounds have an apolar residue. Within one subclass, e.g., the derivatives of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), it has been shown that the distances between the carboxylate group and the amino group, between the nitro group and the carboxylate group, and between the phenyl ring and the amino group are very critical. For example, the optimal spacer between the phenyl ring and the amino group was a propyl group. Changing this spacer to butyl or ethyl led to dramatic reductions in the potency. Also the sidedness of substituents on one of the chiral centers of this spacer led to discrepant results. Whereas a O form of one compound was active, the L form was entirely ineffective.5° Data of this kind have led us to suggest that chloride channel blockers interact with several sites. In the case of NPPB (Fig. 1), these sites may be summarized as follows. (1) An 62 S. H. Ferreira and J. R. Vane, Annu. Rev. Pharmacol. 14, 57 (1974). 63 M. J. Stutts, D. C. Henke, and R. C. Boucher, PJluegersArch. 415, 611 (1990). K. Kunzelmann, H. Pavenstlldt, and R. Greger, PJluegersArch. 415, 172 (1989). 65R. Patarca, O. A. Candia, and P. S. Reinach, Am. J. Physiol. 245, F660 (1983). 66 M. G. Evans, A. Marty, Y. P. Tan, and A. Trautmann, PfluegersArch. 406, 65 (1986).
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PHARMACOLOGICAL AGENTS IN EPITHELIAL TRANSPORT
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anionic site is mandatory; this site is ionized at physiological pH. In all the potent compounds it is a carboxylate group, Other anionic groups such as sulfonyl urea 67 are less effective or have very little effect, as in the case of sulfonates (unpublished). This is remarkable sin~e in the case of loop diuretics of the furosemide type, the comparable site can be any of these anionic groups; (2) the amino "bridge" is necessary; it cannot be replaced by oxygen, phosphorus, or carbonS°; (3) an m-nitro substitution or a p-chloro substitution on the benzoate moiety increases potency; (4) finally, an apolar site of interaction is required. Further d / ~ s on charge effects and on optimal distances have been summarized r ~ . 5° Table II summarizes several chloride channel blockers and compares them to closely related blockers of the Na+/2C1-/K + carder. 3 It is apparent from this figure that minute changes in the molecule shift the affinity of one given structure from the chloride channel to the Na+/2C1-/K + carder. A general principle seems to be that the blockers of the Na+/2C1-/K + carrier contain a sulfonamide group or a pyridine nitrogen where the blockers of the chloride channel contain a nitro substitution. I have already stated above that the anionic group is c o m m o n to both classes of substances. Also the amino nitrogen is c o m m o n to both classes. Finally, the apolar residue also appears essential in both kinds of compounds. However, the special restrictions on this group are more marked for the chloride channel blockers. This similarity of blockers for two very distinct chloride permeation pathways stresses that the respective membrane proteins may have molecular similarities as expressed by their "receptor" sites for these blockers. It would not be surprising at this stage if certain amino acid sequences in both membrane proteins would show a high degree of homology. Nevertheless, I wish to emphasize that such an assumption is very indirect and speculative. 67 M. Wittner, A. Di Stefano, P. Wangemann, J. Delarge, J. F. Liegeois, and R. Greger, PfluegersArch. 408, 54 (1987).
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Several of the above blockers have meanwhile been tested in other epithelia. Most of this work is still preliminary. However, there is evidence that these compounds act in a variety of chloride channels in, e.g., rat thick ascending limb,32 frog skin (W. Nagel, personal communication), pancreatic duct,4 sweat gland duct, 6s rectal gland of the dogfish (Squalus acanthias), 69 collecting duct, 7° colonic carcinoma cells,4° rabbit colon (unpublished from our laboratory), and airway epithelial cells. 64 When compared to data obtained from the intact rabbit thick ascending limb,5° it is important to note that the sensitivity to a given blocker may be quite different. For example, in sweat duct or in frog skin the compound 5chlorodiphenylamine-2-carboxylic acid was far more potent than NPPB, the opposite was true for the thick ascending limb of the loop of Henle. This heterogeneity in structure-activity relations for the chloride channel contrasts to the monotonous sequences for the blockers of the Na+/2CI-/ K + carder, where generally the sequence bumetanide > piretanide > furosemide holds true for any tissue possessing this carder system,s,7~ This may indicate that there is nothing like "the epithelial chloride channel" but that chloride channels show differences in the various epithelia, and these differences may also express themselves in the sensitivity to Mockers. The mechanism of chloride channel blocking by these compounds is now being studied in the rectal gland, a7 in colonic carcinoma cells, 12'40'41 and in airway epithelial cells. ~4 The present data indicate that the blockers act from ouside the channel but not from the cytosolic side. The evidence for this is threefold: (1) In inside-out patches the added Mocker acts only with some delay, and also the recovery after removal of the blocker is delayed. We have interpreted these findings in the following way. a7 The blocker must permeate the membrane to reach the channel from the inside of the patch pipet. Also, after removing the blocker from the bulk solution, the blocker must diffuse out of the pipet before the blocking effect is reversed; (2) more direct proof comes from the finding that the effect of a low dose of NPPB, unlike in inside-out patches, was instantaneous in outside-out membrane patches of colonic carcinoma cells72; (3) recently we examined derivatives of NPPB coupled to a macromolecular (5 kDa) 6s j. Bijman, H. C. Englert, H. J. Lang, R. Crr~er, and E. FrOmter, Pfluegers Arch. 408, 511 (1987). 69 R. Greger and E. Schlatter, Pfluegers Arch. 402, 364 (1984). 7oK. Tago, D. H. Warden, V. L. Schuster, and J. B. Stokes, Am. J. Physiol. 251, F1109 (1986). ~ R. Greger and E. Schlatter, Klin. Wochenschr. 61, 1019 (1983). 72 R. Greger, L. Gerlach, and K. Kunzelmann, in "SKF Symposium: Ion Transport" (T. Rink, ed.). Academic Press, San Diego, California, in press, 1989.
[45]
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residue and found that these compounds block only in outside-out patches (unpublished observations). We have also examined the kinetics of the chloride channel during the blocking process, and we find that low doses of a blocker (< 10-6 mol/liter) lead to a reduction in the open probability, mainly by a reduction in the number of long lasting (time constant 5-20 msec) open states. Thus, the channel transits through a rapidly flickering state before it appears completely closed at higher blocker concentrations. Given the complexity of the kinetics of chloride channels it appears premature to deduce a certain model of action for the blockers from these data. In a recent study in vesicles prepared from renal cortex a variety of putative blockers of chloride conductance w a s t e s t e d , 73 and it was found that NPPB was effective in the micromolar concentration range, but that an entirely unrelated compound derived from phenoxyacetic acid55 was at least equally effective. This latter compound was used as an affinity probe to isolate the chloride channel protein. Similarly, the ability of stilbenes to block the chloride channel of airway cellsTM was used to isolate a SITS-binding protein which is supposed to correspond to the chloride channel. 75 Conclusion
Chloride channel blockers comprise very heterogeneous compounds. Thc antagonists of the neuronal chloride channels have nothing in common with, e.g., the blockers for the epithelial chloride channel. On the other hand, fairly simple structures such as the diphcnylaminc-2-carboxylares and the related arylaminobenzoatcs block chloride channels in various tissues. These compounds arc closely related to the known blockers of other chloride-transporting pathways such as the band 3 protein (e.g., in red blood cells) and the Na+/2C1-/K + carrier system. The purification and isolation of the membrane proteins transporting chloride is a very active field of current research. It is expected that a few years from now the amino acid sequences of most of these transport proteins will be available, and we will understand more about the mechanisms of blocker interaction. The blockers available now will probably be powerful tools in the purification process. 73 D. W. Landry, M. Reitman, E. J. Cragoe, and Q. AI-Awqati, J. Gen. Physiol. 90, 779 (1987). 74 R. J. Bridges, R. T. Worrell, R. A. FrizzeU, and D. J. Benos, Am. J. Physiol. 256, C902 (1989). 75 D. J. Benos, Proc. Forefront Symp. NephroL, 3rd p. 62 (1989).
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Acknowledgments Work from the author's laboratory cited in this chapter has been supported by Deutsche Forschungsgemeinschaft (Gr 480/8) and by the "Kommission der Europaischen Gemeinschaften" [ST2J-0095-2-D (CD)]. The secretarial assistance by Mrs. E. Viereck is gratefully acknowledged.
CHLORIDE CHANNEL BLOCKERS
[45] C h l o r i d e
Channel
793
Blockers
By RAINER GREGER
After a brief introduction into the chloride-transporting membrane proteins, this chapter will focus on a new class of chloride channel blockers. It will be shown that these substances belong to a larger family of agents, of which so-caUed loop diuretics are just one member. Introduction This contribution restricts itself to the blocking of chloride channels and not to chloride permeation in general. The ability to distinguish between permeability in general and permeation through a channel depends on the methods used. If radiolabeled chloride is the measured signal, all permeation pathways for chloride existing in the membrane under study will contribute to the signal. If the chloride flux is measured electrically, only systems transporting chloride with its negative charge will be monitored. It should be clear that this latter chloride flux should only be called "electrogenic," and it cannot, a priori, be equated with the flux of chloride through ionic channels. Fortunately, this distinction appears puristic from a practical point of view since only very few rheogenic chloridetransporting carrier systems have been described thus far, whereas chloride channels are present in many different cell types. In the last 10 years or so an identification of chloride channels has become possible by the patchclamp technique.t Still, however, most of our knowledge about blockers of chloride current comes from electrophysiological studies where macroscopic currents or voltages have been measured. In this chapter only chloride transport systems and, more specifically, chloride channels in a few well-defined preparations, mostly of vertebrate offgin, are considered. Chloride channel blockers with very little molecular modification, or generally at higher concentrations, also inhibit other chloride-transporting proteins. This finding has two ramifications on which I will expand. (1) Blockers are usually far less specific than one wishes them to be, and much caution is needed to deduce the existence of a transport system solely on the basis of inhibitor interaction. This consideration, as trivial as it is, is still ignored frequently, and the simple rule that a pharmacological effect must always be looked at on the basis of dose-response curves is neglected in many reports. (2) The fact that a blocker interferes with different affinit B. S a k m a n n a n d E. Neher, "Single-Channel Recording," P l e n u m , N e w York, 1983.
METHODS IN ENZYMOLOGY, VOL. 191
Copyright© 1990by AcademicPress,Inc. All rightsof reproduction in any form reserved.
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PHARMACOLOGICALAGENTS IN EPITHELIAL TRANSPORT
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ties with several chloride-transporting epithelia may also indicate that the transport proteins, even though they may appear functionally different, may share in common some part(s) of the molecule. Therefore, I will briefly describe various chloride transport systems. The bicarbonate/chloride exchange system is present in the red cell membrane in the form of the band 3 protein (for a review, see Passow2). This system or modifications of this system are found in many cells, and may serve pH regulation, 3 bicarbonate transport (for a review, see Novak and Greger4), or chloride transport. 5 Another, recently detected6 chloride transport system is the Na+/2C1-/K + carrier. This system is present in many polar and apolar cells (for a review see Ellory et aL7). It is responsible for the cellular uptake of chloride. If present in epithelia, s it serves the transepithelial transport of NaCI, and is usually found together with chloride channels on the opposite cell membranes. 9 Historically, this Na+/2CI-/K ÷ carrier system is remarkable since blockers for this system, the loop diuretics of furosemide type, had been known for more than 20 years before the system itself was recognized. In fact, the loop diuretic-sensitive transport system was long believed to be an energy-dependent chloride pump. Whereas it is clear now that what had been believed to be a primary pump sensitive to furosemide-like substances turned out to be a secondarily active chloride carrier, chloride pumps are still postulated for insect epithelia) ° Another chloride-dependent carrier system has been recently detected, namely a chloride/formate exchange.11
G e n e r a l P r o p e r t i e s of Chloride C h a n n e l s The introduction of the patch-clamp technique has now made it possible to study individual chloride channels, to measure their conductances, permselectivities, opening kinetics, activation mechanisms, and the mechanism of action of inhibitors (for a review, see GOgelein12). Models for the 2 H. Passow,Rev. Physiol. Biochem. Pharmacol. 103, 61 (1986). a R. C. Thomas, Curt. Top. Membr. Transp. 13, (1980). 4 I. Novak and R. Greger, Pfluegers Arch. 411, 546 (1988). 5C. M. Liedtkeand U. Hopfer,Am. £ Physiol. 242, G272 (1982). P. Geck, C. Pietrzyk, B.-C. Burekhardt, B. Pfeiffer,and E. Heinz, Biochim. Biophys. Acta 600, 432 (1980). 7j. C. Ellory, P. B. Dunham, P. J. Logue, and G. W. Stewart,Philos. Trans. R. Soc. London, B 299, 483 (1982). s R. Greger, PhysioL Rev. 65, 760 (1985). 9 R. Greger, E. Sehlatter, and H. G0gelein, News in Physiol. Sci. 1, 134 (1986). ~oG. A. Gereneser, in "Chloride Transport Couplingin BiologicalMembranesand Epithelia" (G. A. Gereneser, ed.), p. 183.Elsevier,Amsterdam, 1984. H L. Schild, G. Giebiseh, L. Karniski, and P. S. Aronson, PfuegersArch. 4117,156 (1986). ~2H. Grgelein, Biochim. Biophys. Acta 947, 521 (1988).
[45]
CHLORIDE CHANNEL BLOCKERS
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channels have been derived from such data. At the same time, ligands are used to purify certain chloride channel proteins and to sequence their amino acids. This area of research has exploded during the last few years. I will restrict myself to summarizing the published work on single channels merely in table form. Arbitrarily, at least at this point, the chloride channels may be divided into those of neuronal origin, those present in smooth and striated muscle, and those found in the various epithelia. As is apparent from Table I, 13-42 it is difficult to find common denominators for the various channels. For example, the single-channel slope conductance varies between less than 1 and 500 pS. Even if one considers that the composition of the bathing solutions may have been different, this range appears surprising. One possible clue to the origin of such a wide range may be the fact that chloride channels appear in clusters within one patch. Such clusters can be taken as subunits of one single channel, which then would have a rather large unitary conductance. Alternatively, clusters t3 D. G. Owen, M. Segal, and J. L. Barker, Nature (London) 311, 567 (1984). t40. P. Hamill, J. Bormann, and B. Sakmann, Nature (London) 305, 805 (1983). t~ R. N. McBarney, S. M. Smith, and R. Zorec, J. Physiol. (London) 367, 68P (1985). ~6A. J. Martin and M. R. Gold, Biophys. J. 41, 62 (1983). t7 V. I. Geletyuk and V. N. Kazachenko, J. Membr. Biol. 86, 9 (1985). t8 D. Chesnoy-Marchais and M. G. Evans, J. Physiol. (London) 357, 64P (1984). ~9M. M. White and C. Miller, J. Biol. Chem. 254, 10161 (1979). 2op. T. A. Gray, S. Bevan, and J. M. Ritchie, Proc. R. Soc. London, B 221, 395 (1984). 2~p. T. A. Gray and J. M. Ritchie, Proc. R. Soc. London, B 228, 267 (1986). 22S. S. Kolesnikov, A. L. Lyubatsky, and E. E. Fesenko, Vision Res. 24, 1295 (1984). 23A. L. Blatz and K. L. Magleby, Biophys. J. 43, 237 (1983). 24A. L. Blatz and K. L. Magleby, Biophys. J. 47, 119 (1985). 25W. Schwarze and H.-A. Kolb, PfluegersArch. 402, 281 (1984). 26A. Coulombe and H. Duclohier, J. Physiol. (London) 350, 52P (1984). 27 R. Shoemaker, J. Naftel, and J. Farley, Biophys. J. 47, 465a (1985). 2s G. P. H. Young, J. D.-E. Young, A. K. Deshpande, M. Goldstein, S. S. Koide, and Z. A. Cohn, Proc. NatL Acad. Sci. U.S.A. 81, 5155 (1984). 29D. J. Nelson, J. M. Tang, and L. G. Palmer, J. Membr. Biol. 80, 81 (1984). 3oH. A. Kolb, C. D. A. Brown, and H. Murer, PfluegersArch. 403, 262 (1985). 31 R. Greger, Physiol. Aktuel. 2, 47 (1986). 32R. Greger, M. Bleich, and E. Schlatter, Renal Physiol. Biochem. 13, 37-50 (1990). 33H. GOgeleinand R. Greger, PfluegersArch. 407 (Suppl. 2), S142 (1986). W. Hanrahan, W. P. Alles, and S. A. Lewis, £ Gen. Physiol. 84, 30a (1984). 3s A. Marty, Y. P. Tan, and A. Trautmann, J. Physiol. (London) 357, 293 (1984). 36I. Findlay and O. H. Petersen, Pfluegers Arch. 403, 328 (1985). 37R. Grager, E. Schlatter, and H. GOgelein,Pfluegers Arch. 409, 114 (1987). 3s H. G6gelein, E. Schlatter, and R. Greger, Pfluegers Arch. 409, 122 (1987). 39M. J. Welsh, PfluegersArch. 407 (Suppl. 2), S116 (1986). 4oj. p. Hayslett, H. G6gelein, K. Kunz¢lmann, and R. Greger, Pfluegers Arch. 410, 487 (1987). 4t R. Greger and K. Kunzelmann, in "Epithelial Secretion of Water and Electrolytes" (J. A. Young and P. D. Wong, eds.). Springer-Verlag, New York, 1989. 42M. E. Krouse, G. T. Schneider, and P. W. Gage, Nature (London) 319, 58 (1986).
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are interpreted as individual channels, which then leads to rather small values for the unitary conductance. According to accepted theory, a distinction between both alternative explanations can be made on the basis of whether the single conductance events (or sublevels) are dependent on or independent of each other as tested by statistical methods. In case of independence of events, the term subunit would be misleading if it was not qualified as independent subunit. If the conductance events are dependent on each other, independent single channels are excluded and the term subunit is appropriate. The situation may be even more complex if one looks closely at original recordings. Frequently, substates of differing current amplitude are observed, and even one individual chloride channel may vary its slope conductance to some extent as the experiment proceeds. To make it even worse, in one patch several different chloride channels can be found. This has been recognized in myotubes,23~ and in epithelia. ~2,aS,a7,3sUsually, large and small channels are distinguished, but if there were no additional criteria for distinction, the slope conductance alone would clearly not be sufficient. Finally, it should not be ignored that the patch-clamp technique for the recording of single ionic channels has its restrictions. It will "see" a channel only if it is open for some limited time and at some limited current level. Our routine conditions (signal-to-noise ratio for a filter frequency of 0 . 5 - l kHz) would set the limit such that a channel will be seen only if it is open at least some 0.2-0.5 msec and if it has a conductance above 5 pS. Thus, in single-channel recordings, we will ignore any very small chloride channel whether it was physiologically relevant or not. It should be clear, however, that even very small channels can be found in whole cell recordings. To summarize this issue, it seems very dangerous to classify chloride channels according to their conductance. Inherent pitfalls include the substate and subunit dilemma, and, probably even more serious, the variability of the conductance for otherwise identical channels. A similar dilemma in classification of chloride channels is apparent if one considers the heterogeneity of the voltage dependence. Three patterns are found with almost equal frequency: (l) increase in open probability by depolarization, (2) increase in open probability by hyperpolarization, and (3) bell-shaped curves with the maximum around 0 mV clamp potential. These different patterns cannot be ascribed to the three subclasses of channels chosen for this table. In fact, for neuronal chloride channels all three patterns have been reported. For the epithelial chloride channels it seems to be the "rule" that depolarization increases the open probability and, in many instances, also the slope conductance. Where determined, the listed channels are clearly anion selective, and large anions are usually excluded. At least in some types of channels I- and
[45]
CHLORIDE CHANNEL BLOCKERS
799
to a smaller extent Br- are excluded. Chloride channels may be regulated by Ca 2+, but the calcium dependence is also heterogeneous and ranges from Ca 2+ dependent to Ca2+ inhibited. However, as with selectivity, too few data have been reported to arrive at any firm conclusion. To summarize Table I, I should like to conclude that chloride channels in the various cells show a large variability. In this respect, other ionic channels, e.g., the acetylcholine receptor, the Na + channels, and the K + channels, seem to be more homogeneous. As devastating as the data in Table I may be, I am optimistic that subclasses of channels are emerging with far less heterogeneity, as, for example, the various chloride channels in secretory epithelia. Most research groups would agree that the cAMP-dependent chloride channels present in these epithelia have a narrow conductance range (around 40-50 pS), a similar selectivity, and a comparable voltage dependence. Chloride C h a n n e l Blockers According to their targets, the various agents may be grouped into substances interfering with the 7-aminobutyfic acid (GABA) and glycine receptor chloride channels, and agents which interfere with chloride channels in muscle membrane, apolar cells such as the red blood cell, and in epithelial cells. This type of classification is not at all satisfactory since, on the one hand, some agents appear to act in several of the above-described channel types. On the other hand, the representatives for one group of blockers act quite differently. Some may interfere with the activation of the channel, e.g., GABA antagonists, others may act at the chloride-binding site, and yet others are not at all defined in their mode of action. Therefore, this chapter will list the different blockers, provide reference to pertinent recent work, but will not go into details of the mechanism of blocking. For one newly detected group of agents, namely the arylaminobenzoates, sufficient data are available to discuss some hypothetical views of the mechanism of interaction.
Agents Acting at the Neuronal Chloride Channels Well-studied examples are the GABA receptor and the glycine receptor chloride channels. GABA binding gates the former channel and that of glycine the latter. 14 The GABA interaction is modified by diazepam and related agonists (central type of receptor) such that the GABA effect is enhanced. Diazepam antagonists have the opposite effect and, therefore, reduce the GABA effect on the chloride current. These substances then act as convulsants. It is interesting to note that shortly after their detection in
800
PHARMACOLOGICAL AGENTS IN EPITHELIAL TRANSPORT
[45]
the brain, peripheral diazepam receptors were also found. The function of these receptors, e.g., in the nephron43 is still obscure. This appears puzzling as the benzodiazepam binding coincides with the site of active NaCl reabsorption in the nephron. This has prompted us to examine several of these agonists and antagonists (kindly provided by Dr. P. Skolnick) in isolated perfused thick ascending limb segments of rabbit and mouse kidney. We are unable to demonstrate an effect on active NaC1 reabsorption (unpublished observations). Other, and chemically not at all related, substances have been reported as inhibitors of the GABA-activated chloride channel: pentylenetetrazole,~ picrotoxin and bicuculline,45 and bicyclic phosphates and several insecticides. ~ The glycine-operated chloride channel is inhibited by strychnine and related compounds. In fact, these antagonists have been used to purify this channel protein. 47
Agents Acting at the Muscular Chloride Channel No experiments are available at the level of single chloride channels (patch-clamp analysis). Therefore, the data on putative blockers of chloride fluxes may reflect the properties of systems other than chloride channels. In the striated barnacle muscle cAMP-stimulated chloride fluxes were inhibited by rather low doses of sulfonic acid stilbene derivatives4S: 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This result documents the similarity of this chloride transport system in muscle cell membrane to that in the red blood cell (vide infra). Also, furosemide in a rather high dose (0.6 mmol/liter) was effective as a reversible inhibitor in this muscle preparation. As will be pointed out later, it is not pertinent, and may even be incorrect, to conclude from this finding that chloride transport occurred via the Na+/2C1-/K+ cotransport system. A recent review49 summarizes the work with chloride channel blockers in striated muscle and subdivides the agents into four groups: (1) polycyclics, with anthracene-9-carboxylate as one example, (2) benzoates, with furosemide as an example, (3) phenoxyacetates, with ethaerynic acid as 43 D. Butlen, FEBSLett. 169, 138 (1984). 44 B. A. Weissmann, J. Cott, D. Hommer, R. Quirion, S. Paul, and P. Skolnick, in "Benzodiazepine Recognition Site Ligands: Biochemistry and Pharmacology" (G. Biggio and E. Costa, eds.), p. 139. Raven, New York, 1983. 45 K. G. Thampy and E. M. Barnes, Jr., J. Biol. Chem. 259, 1753 (1984). 46 j. R. Bloomquist and D. M. Soderlund, Biochem. Biophys. Res. Commun. 133, 37 (1985). 47 D. Graham, F. Pfeiffer, and H. Betz, Eur. J. Biochem. 131, 519 (1983). 48j. M. Russel and M. S. Brodwick, J. Gen. Physiol. 78, 499 (1981). 49A. H. Bretag, Physiol. Rev. 67, 618 (1987).
[45]
CHLORIDE CHANNEL BLOCKERS
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one example, and (4) sulfonates and sulfamides, with disulfonic stilbenes as an example. It is difficult to accept the first three groups as different entities, since it will be shown below that these agents have far more in common than may be apparent at first glance. The methodology, as it has been applied to date, does not permit any conclusion as to how these substances reduce chloride fluxes or chloride conductance. However, recent studies with these substances in epithelial chloride channels5° (cf. next section) may also be used to extrapolate these data to the muscle chloride channel. In the smooth muscle cell most evidence favors the view that cytosolic chloride activity is above Nernstian equilibrium. 51 Many data are available on anion replacements on the extracellular compartment and their effect on membrane voltage, as are many data on disulfonate stilbene as well as on furosemide effects. Little, however, is known on the properties of the respective chloride channels. 27 It has been argued that SITS or DIDS, in analogy to their effects in Torpedo electric organ chloride channel, 52 may also interfere with chloride channels in smooth muscle cells. Also, the effects of organic "nitro" compounds, such as nitroglycerin, have been ascribed to the interference of these compounds with chloride channels. 51
Agents Acting in Apolar Cells One specific, but obviously very complex, chloride transport system is that of the red blood cell. There, the vast majority of the chloride flux occurs via the band 3 protein. 2 However, chloride channels are also present in these cells. A recent survey has compared several classes of agents in their ability to block the anion shift via the band 3 protein and the chloride movement through single chloride channels. 53 It was concluded that both routes of chloride movement take the same pathway, namely, via the band 3 protein. In other words, this protein may perform electroneutral anion exchange (quantitatively most important), and may also permit the channeling of chloride ions. From this point of view, older data using furosemide 2 and flufenamic aci@4 may be looked at from a new perspective. Flufenamic acid and related substances belong to the group of chloride channel blockers, cf below. The band 3 protein may thus bind blockers of three different classes: the sulfonic acid stilbene derivatives, compounds related to furosemide, and compounds related of the arylaminobenzoate so p. Wangemann, M. Wittner, A. Di Stefano, H. C. Englert, H. J. Lang,E. Schlatter, and R. Greger, Pfluegers Arch, 407 (Suppl. 2), S128 (1986). 5~ V. A. W. Kreye and F. W. Ziegler, Adv. Microcirc. 11, 114 (1982). 52C. Miller and M. M. White, Proc. Natl. Acad. Sci. U.S.A. 81, 2772 (1984). 53 W. Schwarz and H. Passow, Proc. Int. Union Physiol. Sci. 30th Congr. 16, 544 (1986). 54J.-L. Cousin and R. Motais, Biochim. Biophys. Acta 687, 156 (1982).
802
PHARMACOLOGICAL AGENTS IN EPITHELIAL TRANSPORT
[45]
class. It is important to realize that the affinities to these three classes of substances are quite different from those needed in other preparations. Micromolar concentrations are needed to block the band 3 protein by sulfonic acid stilbenes, yet much higher concentrations are usually required in other systems. Conversely, the concentrations needed of furosemide and even more so of bumetanide are much higher for the band 3 protein when compared to that needed to interfere with the Na+/2CI-/K + cartier system,s Similarly, rather high concentrations of arylaminobenzoates are necessary to inhibit the band 3 protein. A great deal of effort has been directed toward the pharmacology of glial cell volume regulation, and it was recognized some 4 years ~tgo that several transport systems in which chloride participates are probably responsible for the glial volume increase, which in turn may be the pathophysiological basis of traumatic brain edema. In a recent study, several classes of substances have been tested in brain slices and it was found that the most effective inhibitors were derived from the phenoxyacetic acid group. Specifically (2,3,9,9a-tetrahydro-3-oxo-9ot-substituted-lH-fluoren7-yl)oxyalkanoic acids were effective in doses as low as 10-12 mol/liter. However, the authors 55 concluded only that this class of inhibitors blocks C1- uptake and have no evidence that this uptake is via chloride channels.
Agents Acting in Epithelia Soon after the recognition of the existence of chloride channels in epithelia such as the thick ascending limb, the colon, and the trachea, a search for agents blocking these channels was initiated in our laboratory. The focus was on anthracene-9-carboxylate and diphenylamine-2-carboxylate since these two substances, previously used in the striated muscle chloride channel 5n and in the red blood cell,54 showed some inhibitory activity on the chloride conductive pathways in the thick ascending limb 57 and in the amphibian diluting segment.5s Our survey covered a broad spectrum of substances and used a more systematic approach for the substances showing strong activity. The bioassay used in this survey, namely, the in vitro perfused thick ascending limb of the loop of Henle, has the great advantage that chloride conductance is present only at the blood side of the cell, whereas the Na+/2CI-/K + carrier is present only in the 55E. J. Cragoe, Jr., O. W. Woltersdoff, Jr., N. P. Gould, A. M. Pietruszkiewie-z, C. Ziegler, Y. Sakurai, G. E. Stokker, P. S. Anderson, R. S. Bourke, H. K. Kimelberg, L. R. Nelson, K. D. Barron, J. R. Rose, D. Szarowski, A. J. Popp, and J. B. Waldman, J. Med. Chem. 29, 825 (1986). 56 p. T. Palade and R. L. Barchi, J. Gen. Physiol. 69, 879 (1977). 57A. Di Stefano, M. Wittner, E. Schlatter, H. J. l_ang, H. Englert, and R. Greger, Pfluegers Arch. 405 (Suppl. 1), $95 (1985). 5s H. Oberleithner, M. Ritter, F. Lang, and W. Guggino, Pfluegers Arch. 398, 172 (1983).
[45]
CHLORIDE CHANNEL BLOCKERS
803
lumenal membrane) Loop diuretics of the furosemide type interfere with this carder system but have little effect on chloride channels. A simple experiment leads to this distinction: whereas loop diuretics inhibit the active transport of chloride and sodium instantaneously when added to the lumenal perfusate, they are devoid of effect when added to the basolateral perfusate. Conversely, blockers of the arylaminobenzoate type, as they are listed in Table II, block the active transport of chloride at low concentrations when added to the basolateral perfusate but not when added at the same concentration to the lumenal perfusate. Nevertheless, if the furosemide concentration on the basolateral cell pole is increased to very high concentrations of 10-a tool/liter or more, some inhibition will also be observed. Similarly, if the chloride channel blocker is added to the lumenal perfusate at very high concentration, it may also exert an effect from this side. These effects may be due to a diffusion of furosemide or the chloride channel blocker to the other cell membrane or to effects other than that on the Na+/2C1-/K + carder or on the chloride channel. The first explanation becomes more likely the higher the lipid solubility of the tested compound is. In case of furosemide and related compounds, the lipid solubility at physiologic pH is rather low, but for the chloride channel blockers the opposite is true. Some 30-80% distribute into the lipid phase (e.g., CH2CI2) at pH 7.4. It is not surprising then that these substances also act usually at 10-100 times higher concentrations from the lumenal cell side. 5° The second explanation given above comprises a heterogeneous group of possible effects. Of course, it must be considered that one individual substance may not only inhibit chloride channels, but may also interact with the Na+/2C1-/K + carrier. We have several examples available. 59,6° It should be clear that preparations other than the intact epithelium, e.g., the isolated membrane patch, must be used to prove this potential effect of an Na+/2C1-/K + carrier blocker on the chloride channel. Another potential complication comes from the fact that the tested compound may interfere with other systems. For high concentrations of furosemide and related compounds, an interference with carbonic anhydrase has been shown.61 It should also be noted that the loop diuretics of furosemide type have originated from studies with carbonic anhydrase inhibitors. Furthermore, lipophilic anions will also find access to the mitochondria and will short circuit pH gradients across thinner mitochondrial membrane, thus inter59 R. Greger, P. Wangemann, M. Wittner, A. Di Stefano, H. J. Lang, and H. C. Englert, in "International Conference on Diuretics, Sorento, 1986" (T. Andreucci, ed.), p. 33. Nijhoff, Boston, Massachusetts, 1987. 6o R. Greger, H. J. Lang, H. C. Englert, and P. Wangemann, in "Diuretics" (J. Puschett, ed.), p. 131. Elsevier, New York, 1987. 6~ T. H. Maren, "Renal Physiology: Men and Ideas." Am. Physiol. Sot., Bethesda, Maryland, 1987.
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feting with mitochondrial function. This putative mechanism may be responsible for the poorly reversible inhibitory effect seen with arylaminobenzoates at high concentrations. This effect is usually followed by a cell depolarization. 5° The chloride channel blockers of the arylaminobenzoate type are also known to block cyclooxygenase,~2 and this may generate a dilemma in tissues in which the chloride conductance is increased by prostaglandins. Hence, the reduction in chloride conductance observed in airway epithelial cells after the addition of very high concentrations of diphenylamine-2-carboxylate39 may, in part, be caused by cyclooxygenase inhibition. 63 From our studies we have determined that furosemide is not a chloride channel blocker, u and this contrasts to other studies on the cornea65 and on the tear gland, u It should be noted, however, that in the intact preparation, furosemide will lead to a reduction of the chloride conductance simply because it reduces cytosolic chloride activity,s In isolated chloride channels, we have never observed any effect of furosemide.37,u Table III summarizes several potent chloride channel blockers as documented by their inhibitory effect on the chloride conductance in isolated perfused thick ascending limbs. Some of these compounds have also been tested in isolated chloride channels of the rectal gland, a7 in airway epithelial cells, u in the thick ascending limb of the loop of Henle, 32 and in colonic carcinoma cells?° It is apparent that all listed compounds possess several common features. All are anionic at physiological pH. The anionic group is a carboxylate. All compounds possess an amino group, and all compounds have an apolar residue. Within one subclass, e.g., the derivatives of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), it has been shown that the distances between the carboxylate group and the amino group, between the nitro group and the carboxylate group, and between the phenyl ring and the amino group are very critical. For example, the optimal spacer between the phenyl ring and the amino group was a propyl group. Changing this spacer to butyl or ethyl led to dramatic reductions in the potency. Also the sidedness of substituents on one of the chiral centers of this spacer led to discrepant results. Whereas a O form of one compound was active, the L form was entirely ineffective.5° Data of this kind have led us to suggest that chloride channel blockers interact with several sites. In the case of NPPB (Fig. 1), these sites may be summarized as follows. (1) An 62 S. H. Ferreira and J. R. Vane, Annu. Rev. Pharmacol. 14, 57 (1974). 63 M. J. Stutts, D. C. Henke, and R. C. Boucher, PJluegersArch. 415, 611 (1990). K. Kunzelmann, H. Pavenstlldt, and R. Greger, PJluegersArch. 415, 172 (1989). 65R. Patarca, O. A. Candia, and P. S. Reinach, Am. J. Physiol. 245, F660 (1983). 66 M. G. Evans, A. Marty, Y. P. Tan, and A. Trautmann, PfluegersArch. 406, 65 (1986).
806
PHARMACOLOGICAL AGENTS IN EPITHELIAL TRANSPORT
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anionic group
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anionic site is mandatory; this site is ionized at physiological pH. In all the potent compounds it is a carboxylate group, Other anionic groups such as sulfonyl urea 67 are less effective or have very little effect, as in the case of sulfonates (unpublished). This is remarkable sin~e in the case of loop diuretics of the furosemide type, the comparable site can be any of these anionic groups; (2) the amino "bridge" is necessary; it cannot be replaced by oxygen, phosphorus, or carbonS°; (3) an m-nitro substitution or a p-chloro substitution on the benzoate moiety increases potency; (4) finally, an apolar site of interaction is required. Further d / ~ s on charge effects and on optimal distances have been summarized r ~ . 5° Table II summarizes several chloride channel blockers and compares them to closely related blockers of the Na+/2C1-/K + carder. 3 It is apparent from this figure that minute changes in the molecule shift the affinity of one given structure from the chloride channel to the Na+/2C1-/K + carder. A general principle seems to be that the blockers of the Na+/2C1-/K + carrier contain a sulfonamide group or a pyridine nitrogen where the blockers of the chloride channel contain a nitro substitution. I have already stated above that the anionic group is c o m m o n to both classes of substances. Also the amino nitrogen is c o m m o n to both classes. Finally, the apolar residue also appears essential in both kinds of compounds. However, the special restrictions on this group are more marked for the chloride channel blockers. This similarity of blockers for two very distinct chloride permeation pathways stresses that the respective membrane proteins may have molecular similarities as expressed by their "receptor" sites for these blockers. It would not be surprising at this stage if certain amino acid sequences in both membrane proteins would show a high degree of homology. Nevertheless, I wish to emphasize that such an assumption is very indirect and speculative. 67 M. Wittner, A. Di Stefano, P. Wangemann, J. Delarge, J. F. Liegeois, and R. Greger, PfluegersArch. 408, 54 (1987).
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Several of the above blockers have meanwhile been tested in other epithelia. Most of this work is still preliminary. However, there is evidence that these compounds act in a variety of chloride channels in, e.g., rat thick ascending limb,32 frog skin (W. Nagel, personal communication), pancreatic duct,4 sweat gland duct, 6s rectal gland of the dogfish (Squalus acanthias), 69 collecting duct, 7° colonic carcinoma cells,4° rabbit colon (unpublished from our laboratory), and airway epithelial cells. 64 When compared to data obtained from the intact rabbit thick ascending limb,5° it is important to note that the sensitivity to a given blocker may be quite different. For example, in sweat duct or in frog skin the compound 5chlorodiphenylamine-2-carboxylic acid was far more potent than NPPB, the opposite was true for the thick ascending limb of the loop of Henle. This heterogeneity in structure-activity relations for the chloride channel contrasts to the monotonous sequences for the blockers of the Na+/2CI-/ K + carder, where generally the sequence bumetanide > piretanide > furosemide holds true for any tissue possessing this carder system,s,7~ This may indicate that there is nothing like "the epithelial chloride channel" but that chloride channels show differences in the various epithelia, and these differences may also express themselves in the sensitivity to Mockers. The mechanism of chloride channel blocking by these compounds is now being studied in the rectal gland, a7 in colonic carcinoma cells, 12'40'41 and in airway epithelial cells. ~4 The present data indicate that the blockers act from ouside the channel but not from the cytosolic side. The evidence for this is threefold: (1) In inside-out patches the added Mocker acts only with some delay, and also the recovery after removal of the blocker is delayed. We have interpreted these findings in the following way. a7 The blocker must permeate the membrane to reach the channel from the inside of the patch pipet. Also, after removing the blocker from the bulk solution, the blocker must diffuse out of the pipet before the blocking effect is reversed; (2) more direct proof comes from the finding that the effect of a low dose of NPPB, unlike in inside-out patches, was instantaneous in outside-out membrane patches of colonic carcinoma cells72; (3) recently we examined derivatives of NPPB coupled to a macromolecular (5 kDa) 6s j. Bijman, H. C. Englert, H. J. Lang, R. Crr~er, and E. FrOmter, Pfluegers Arch. 408, 511 (1987). 69 R. Greger and E. Schlatter, Pfluegers Arch. 402, 364 (1984). 7oK. Tago, D. H. Warden, V. L. Schuster, and J. B. Stokes, Am. J. Physiol. 251, F1109 (1986). ~ R. Greger and E. Schlatter, Klin. Wochenschr. 61, 1019 (1983). 72 R. Greger, L. Gerlach, and K. Kunzelmann, in "SKF Symposium: Ion Transport" (T. Rink, ed.). Academic Press, San Diego, California, in press, 1989.
[45]
CHLORIDE CHANNEL BLOCKERS
809
residue and found that these compounds block only in outside-out patches (unpublished observations). We have also examined the kinetics of the chloride channel during the blocking process, and we find that low doses of a blocker (< 10-6 mol/liter) lead to a reduction in the open probability, mainly by a reduction in the number of long lasting (time constant 5-20 msec) open states. Thus, the channel transits through a rapidly flickering state before it appears completely closed at higher blocker concentrations. Given the complexity of the kinetics of chloride channels it appears premature to deduce a certain model of action for the blockers from these data. In a recent study in vesicles prepared from renal cortex a variety of putative blockers of chloride conductance w a s t e s t e d , 73 and it was found that NPPB was effective in the micromolar concentration range, but that an entirely unrelated compound derived from phenoxyacetic acid55 was at least equally effective. This latter compound was used as an affinity probe to isolate the chloride channel protein. Similarly, the ability of stilbenes to block the chloride channel of airway cellsTM was used to isolate a SITS-binding protein which is supposed to correspond to the chloride channel. 75 Conclusion
Chloride channel blockers comprise very heterogeneous compounds. Thc antagonists of the neuronal chloride channels have nothing in common with, e.g., the blockers for the epithelial chloride channel. On the other hand, fairly simple structures such as the diphcnylaminc-2-carboxylares and the related arylaminobenzoatcs block chloride channels in various tissues. These compounds arc closely related to the known blockers of other chloride-transporting pathways such as the band 3 protein (e.g., in red blood cells) and the Na+/2C1-/K + carrier system. The purification and isolation of the membrane proteins transporting chloride is a very active field of current research. It is expected that a few years from now the amino acid sequences of most of these transport proteins will be available, and we will understand more about the mechanisms of blocker interaction. The blockers available now will probably be powerful tools in the purification process. 73 D. W. Landry, M. Reitman, E. J. Cragoe, and Q. AI-Awqati, J. Gen. Physiol. 90, 779 (1987). 74 R. J. Bridges, R. T. Worrell, R. A. FrizzeU, and D. J. Benos, Am. J. Physiol. 256, C902 (1989). 75 D. J. Benos, Proc. Forefront Symp. NephroL, 3rd p. 62 (1989).
810
PHARMACOLOGICAL AGENTS IN EPITHELIAL TRANSPORT
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Acknowledgments Work from the author's laboratory cited in this chapter has been supported by Deutsche Forschungsgemeinschaft (Gr 480/8) and by the "Kommission der Europaischen Gemeinschaften" [ST2J-0095-2-D (CD)]. The secretarial assistance by Mrs. E. Viereck is gratefully acknowledged.
